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BioLegend phycoerythrin pe conjugated anti cd45
TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage <t>(CD45</t> + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P
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1) Product Images from "TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice"

Article Title: TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.08.022

TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage (CD45 + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P
Figure Legend Snippet: TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage (CD45 + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P

Techniques Used: Inhibition, Immunofluorescence, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Cell Culture

TRPA1 inhibition prevents pressure overload-induced macrophage infiltration in cardiac tissues. (A) Immunohistochemical analysis of CD3 and CD68 in heart sections (n = 7, scale bar, 50 μm). (B–C) Flow cytometry analysis of CD45 + cells and T cells (CD45 + CD3 + ) in mice at 4 weeks after TAC surgery (n = 4). *P
Figure Legend Snippet: TRPA1 inhibition prevents pressure overload-induced macrophage infiltration in cardiac tissues. (A) Immunohistochemical analysis of CD3 and CD68 in heart sections (n = 7, scale bar, 50 μm). (B–C) Flow cytometry analysis of CD45 + cells and T cells (CD45 + CD3 + ) in mice at 4 weeks after TAC surgery (n = 4). *P

Techniques Used: Inhibition, Immunohistochemistry, Flow Cytometry, Cytometry, Mouse Assay

2) Product Images from "Interferon-? (IFN-?) suppresses HTLV-1 gene expression and cell cycling, while IFN-? combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells"

Article Title: Interferon-? (IFN-?) suppresses HTLV-1 gene expression and cell cycling, while IFN-? combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells

Journal: Retrovirology

doi: 10.1186/1742-4690-10-52

Effects of IFN-α treatment on HTLV-1 p19 release and viral transcription in various HTLV-1-infected cell lines. A . Expression of HTLV-1 mRNAs ( a ) and proteins ( b , c ) were evaluated by quantitative RT-PCR ( a ), immunoblotting ( b ), and flow cytometry ( c ), respectively, in HTLV-1-infected HUT102, ILT-Hod and ILT-#29 or uninfected Jurkat cell lines. a . The mRNA copy numbers measured by using pX or Gag primers were standardized to those for GAPDH and indicated as the means and standard deviations (SD) of duplicate samples. b . Cell lysates from indicated cell lines were subjected to an immunoblotting assay with antibodies to Tax (40 kDa) and α-Tubulin (50 kDa). The lysates in lanes 5 and 6 were prepared from ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) overnight, respectively. c . Intracellular Tax proteins in permeabilized cells were stained with Alexa Fluor 488-labeled anti-Tax mAb (open histogram) and mouse IgG3 isotype control antibody (closed histogram). The inserted box indicates Gag expression in ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) for 17h. B . HUT102 (top), ILT-Hod (middle) and ILT-#29 (bottom) cells were cultured for 3 days with or without three doses of IFN-α indicated. HTLV-1 p19 concentrations in the supernatants (left) and Gag mRNA levels were measured by ELISA and quantitative RT-PCR, respectively. Data are presented as the means and SD of duplicate samples. C . Frozen stored primary ATL cells were thawed and analyzed for intracellular Tax (top) or Gag (bottom) proteins by flow cytometry immediately (green line) or 24 h after culture with no (black line), 300 IU/ml (red line) or 3000 IU/ml (blue line) of IFN-α in the presence of IL-2 (30 IU/ml). The closed histogram represents samples stained with isotype controls. The mean fluorescence intensity (MFI) of each histogram was indicated in the bar graphs.
Figure Legend Snippet: Effects of IFN-α treatment on HTLV-1 p19 release and viral transcription in various HTLV-1-infected cell lines. A . Expression of HTLV-1 mRNAs ( a ) and proteins ( b , c ) were evaluated by quantitative RT-PCR ( a ), immunoblotting ( b ), and flow cytometry ( c ), respectively, in HTLV-1-infected HUT102, ILT-Hod and ILT-#29 or uninfected Jurkat cell lines. a . The mRNA copy numbers measured by using pX or Gag primers were standardized to those for GAPDH and indicated as the means and standard deviations (SD) of duplicate samples. b . Cell lysates from indicated cell lines were subjected to an immunoblotting assay with antibodies to Tax (40 kDa) and α-Tubulin (50 kDa). The lysates in lanes 5 and 6 were prepared from ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) overnight, respectively. c . Intracellular Tax proteins in permeabilized cells were stained with Alexa Fluor 488-labeled anti-Tax mAb (open histogram) and mouse IgG3 isotype control antibody (closed histogram). The inserted box indicates Gag expression in ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) for 17h. B . HUT102 (top), ILT-Hod (middle) and ILT-#29 (bottom) cells were cultured for 3 days with or without three doses of IFN-α indicated. HTLV-1 p19 concentrations in the supernatants (left) and Gag mRNA levels were measured by ELISA and quantitative RT-PCR, respectively. Data are presented as the means and SD of duplicate samples. C . Frozen stored primary ATL cells were thawed and analyzed for intracellular Tax (top) or Gag (bottom) proteins by flow cytometry immediately (green line) or 24 h after culture with no (black line), 300 IU/ml (red line) or 3000 IU/ml (blue line) of IFN-α in the presence of IL-2 (30 IU/ml). The closed histogram represents samples stained with isotype controls. The mean fluorescence intensity (MFI) of each histogram was indicated in the bar graphs.

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

3) Product Images from "Norovirus P Particle Efficiently Elicits Innate, Humoral and Cellular Immunity"

Article Title: Norovirus P Particle Efficiently Elicits Innate, Humoral and Cellular Immunity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063269

VA387-specific CD4 + T cells in immunized mice. (A) One week after the third immunization, splenocytes were collected and resuspended in RPMI-1640 with 3 µg/ml of brefeldin A and stimulated with 5 µg/ml of VA387 CD4 T cell epitope. Intracellular cytokines of IL-2, IFN-γ and TNF-α were stained as described Figure 4 . (B) Splenocytes were collected from immunized mice and stimulated with VA387-specific CD4 + T cell epitope without brefeldin A. IFN-γ productions in culture supernatant 24 or 48 hours post stimulation were determined by ELISA. ∗ P
Figure Legend Snippet: VA387-specific CD4 + T cells in immunized mice. (A) One week after the third immunization, splenocytes were collected and resuspended in RPMI-1640 with 3 µg/ml of brefeldin A and stimulated with 5 µg/ml of VA387 CD4 T cell epitope. Intracellular cytokines of IL-2, IFN-γ and TNF-α were stained as described Figure 4 . (B) Splenocytes were collected from immunized mice and stimulated with VA387-specific CD4 + T cell epitope without brefeldin A. IFN-γ productions in culture supernatant 24 or 48 hours post stimulation were determined by ELISA. ∗ P

Techniques Used: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

Cytokine production of IL-2, IFN-γ and TNF-α by CD4 + T cells in immunized mice. One week after the third immunization, spleens were collected from mice. Splenocytes were suspended in RPMI-1640 with 3 µg/ml of brefeldin A and were stimulated with PMA and ionomycin for 6 hours. The cells were then stained with surface makers CD3 and CD4, fixed with Fixation Buffer, and then stained with APC-conjugated anti-IL-2 (A), anti-IFN-γ (B) and anti-TNF-α (C) antibodies. ∗ P
Figure Legend Snippet: Cytokine production of IL-2, IFN-γ and TNF-α by CD4 + T cells in immunized mice. One week after the third immunization, spleens were collected from mice. Splenocytes were suspended in RPMI-1640 with 3 µg/ml of brefeldin A and were stimulated with PMA and ionomycin for 6 hours. The cells were then stained with surface makers CD3 and CD4, fixed with Fixation Buffer, and then stained with APC-conjugated anti-IL-2 (A), anti-IFN-γ (B) and anti-TNF-α (C) antibodies. ∗ P

Techniques Used: Mouse Assay, Staining

4) Product Images from "Cross-talk between iNKT cells and CD8 T cells in the spleen requires the IL-4/CCL17 axis for the generation of short-lived effector cells"

Article Title: Cross-talk between iNKT cells and CD8 T cells in the spleen requires the IL-4/CCL17 axis for the generation of short-lived effector cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1913491116

CCR4 and CXCR3 expressions increase over time at the cell surface upon vaccination and play a role in early T cell activation. CD8 + OT-I T cells were isolated, labeled with CFR dye, and adoptively transferred prior vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At different time points, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. ( A ) Representative expression of CXCR3, CCR4, and CD69 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( B and C ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally. ( B ) Six hours after nanovaccine administration, spleens were harvested and CD69 expression on CFR-labeled OT-I T cells were evaluated by flow cytometry. Representative experiment ( Left ). Percentage of inhibition is indicated as the percentage of CD69 + OT-I T cells in blocking antibody-treated cells relative to the percentage of CD69 + OT-I T cells in vehicle-treated cells ( Right ). Pool from 3 independent experiments for CXCR3 blockade and 2 independent experiments for CCR4 blockade. Statistical analysis by t test: * P
Figure Legend Snippet: CCR4 and CXCR3 expressions increase over time at the cell surface upon vaccination and play a role in early T cell activation. CD8 + OT-I T cells were isolated, labeled with CFR dye, and adoptively transferred prior vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At different time points, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. ( A ) Representative expression of CXCR3, CCR4, and CD69 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( B and C ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally. ( B ) Six hours after nanovaccine administration, spleens were harvested and CD69 expression on CFR-labeled OT-I T cells were evaluated by flow cytometry. Representative experiment ( Left ). Percentage of inhibition is indicated as the percentage of CD69 + OT-I T cells in blocking antibody-treated cells relative to the percentage of CD69 + OT-I T cells in vehicle-treated cells ( Right ). Pool from 3 independent experiments for CXCR3 blockade and 2 independent experiments for CCR4 blockade. Statistical analysis by t test: * P

Techniques Used: Activation Assay, Isolation, Labeling, Mouse Assay, Staining, Flow Cytometry, Expressing, Blocking Assay, Injection, Inhibition

NKT cells make long-lasting contacts together with OT-I T cells and DC at late stages of activation. ( A ) Single-cell suspension of splenocytes from CXCR6-GFP mice were stained with CD8, CD3, and NK1.1 antibodies, and Cd1d/α-Galcer dextramer and analyzed by flow cytometry. ( B and F ) CD8 + OT-I T cells were purified, labeled with CFR dye, and adoptively transferred in CXCR6-GFP mice prior to vaccination. One day later, nanovaccines containing OVA and α-Galcer were intravenously administered in CXCR6-GFP mice. At 0, 6, and 24 h, mice were killed, spleens harvested, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining. Cryosections were stained with either anti-CD169 or anti-CD11c antibodies. ( B ) Representative images of the CD169 (red) and CXCR6-GFP (green) costaining at the different time points. (Scale bars, 50 µm.) ( C ) Percentage of the CXCR6 hi cell localized in the WP at the different time points. ( D ) Percentage of the clustering of CXCR6 hi cells at the different time points. ( E ) Percentage of OT-I T cells in close contacts (distance inferior to 3 µm) with CXCR6 hi cells in the MZ/RP or in the WP at the different time points. ( F ) Representative images of OT-I (white), CD11c (red), and CXCR6-GFP (green) costaining at 24 h in the WP. (Scale bar, 10 µm.) ( G ) CD8 + OT-I T cells were purified, labeled with CFR dye and adoptively transferred in CXCR6-GFP mice prior to vaccination. Sixteen hours later, nanovaccines containing OVA and α-Galcer or only α-Galcer were intravenously administered in CXCR6-GFP mice. Six and 24 h later, mice were killed, spleens harvested, and embedded in a low-melting agarose gel. Thick sections of 500 μm were performed using a vibratome. and stained with anti-CD169 and anti-CD11c antibodies. Live imaging was performed using a spinning-disk microscope equipped with a thermostated chamber and perfused at a rate of 0.8 mL/min with medium bubbled with 95% O 2 and 5% CO 2 . OT-I and NKT contacts were evaluated on movies lasting 30 min. ( H ) From analyzed images for the 24-h time point, the positions of NKT and OT-I T cells in the WP were extracted. For each picture, 20 runs of simulation were performed in order to randomly change the location of NKT cells. The closest distance between OT-I and NKT cells was measured for every OT-I T cell and the mean of the closest distance between these 2 cell types was calculated and then compared to the 1 from the original picture. ( I ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally into CXCR6-GFP mice. Then, nanovaccines containing OVA and α-Galcer were intravenously administered, and spleens were harvested 24 h after vaccination, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining with anti-CD169 antibody. NKT cell density (cells/mm 2 ) in the WP was calculated. Results involve at least 2 mice per condition and time point. Statistical analysis by 1-way ANOVA test for C , D , G , and I , and by t test for E and H : * P
Figure Legend Snippet: NKT cells make long-lasting contacts together with OT-I T cells and DC at late stages of activation. ( A ) Single-cell suspension of splenocytes from CXCR6-GFP mice were stained with CD8, CD3, and NK1.1 antibodies, and Cd1d/α-Galcer dextramer and analyzed by flow cytometry. ( B and F ) CD8 + OT-I T cells were purified, labeled with CFR dye, and adoptively transferred in CXCR6-GFP mice prior to vaccination. One day later, nanovaccines containing OVA and α-Galcer were intravenously administered in CXCR6-GFP mice. At 0, 6, and 24 h, mice were killed, spleens harvested, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining. Cryosections were stained with either anti-CD169 or anti-CD11c antibodies. ( B ) Representative images of the CD169 (red) and CXCR6-GFP (green) costaining at the different time points. (Scale bars, 50 µm.) ( C ) Percentage of the CXCR6 hi cell localized in the WP at the different time points. ( D ) Percentage of the clustering of CXCR6 hi cells at the different time points. ( E ) Percentage of OT-I T cells in close contacts (distance inferior to 3 µm) with CXCR6 hi cells in the MZ/RP or in the WP at the different time points. ( F ) Representative images of OT-I (white), CD11c (red), and CXCR6-GFP (green) costaining at 24 h in the WP. (Scale bar, 10 µm.) ( G ) CD8 + OT-I T cells were purified, labeled with CFR dye and adoptively transferred in CXCR6-GFP mice prior to vaccination. Sixteen hours later, nanovaccines containing OVA and α-Galcer or only α-Galcer were intravenously administered in CXCR6-GFP mice. Six and 24 h later, mice were killed, spleens harvested, and embedded in a low-melting agarose gel. Thick sections of 500 μm were performed using a vibratome. and stained with anti-CD169 and anti-CD11c antibodies. Live imaging was performed using a spinning-disk microscope equipped with a thermostated chamber and perfused at a rate of 0.8 mL/min with medium bubbled with 95% O 2 and 5% CO 2 . OT-I and NKT contacts were evaluated on movies lasting 30 min. ( H ) From analyzed images for the 24-h time point, the positions of NKT and OT-I T cells in the WP were extracted. For each picture, 20 runs of simulation were performed in order to randomly change the location of NKT cells. The closest distance between OT-I and NKT cells was measured for every OT-I T cell and the mean of the closest distance between these 2 cell types was calculated and then compared to the 1 from the original picture. ( I ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally into CXCR6-GFP mice. Then, nanovaccines containing OVA and α-Galcer were intravenously administered, and spleens were harvested 24 h after vaccination, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining with anti-CD169 antibody. NKT cell density (cells/mm 2 ) in the WP was calculated. Results involve at least 2 mice per condition and time point. Statistical analysis by 1-way ANOVA test for C , D , G , and I , and by t test for E and H : * P

Techniques Used: Activation Assay, Mouse Assay, Staining, Flow Cytometry, Purification, Labeling, Agarose Gel Electrophoresis, Imaging, Microscopy, Blocking Assay, Injection

IL-4/CCL17 axis promotes the generation of short-lived effector cells. ( A ) nanovaccines containing OVA and α-Galcer or TLR-L were intravenously administered in mice. At different time points, mice were killed, sera were collected, and ELISA for IL-4 was performed. ( B ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At 6 or 24 h, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. Representative expression of pSTAT6 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( C and D ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. One day prior to vaccination, isotype or anti–IL-4 blocking antibodies were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and spleens from the 24-h time point were harvested. ( C ) Expression of CCR4 on OT-I T cells was evaluated by flow cytometry. ( D ) Expression of CCL17 in spleen cryosections (20 µm) was evaluated by confocal microscopy. (Scale bars, 50 µm.) ( E – H ) One-thousand CD8 + OT-I T cells were adoptively transferred prior to vaccination into CD45.1 mice. One day prior to vaccination, isotype, anti-CXCR3 ( E and F ), or anti-CCL17 or IL-4 antibodies ( G and H ) were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and 10 d later spleens were harvested and analyzed by flow cytometry. MPEC were defined as CD127 hi KLGR1 − cells and SLEC as CD127 lo KLGR1 + cells. Pool from 2 independent experiments with at least 5 mice per condition. Statistical analysis by 1-way ANOVA test for A and t test for C – H : * P
Figure Legend Snippet: IL-4/CCL17 axis promotes the generation of short-lived effector cells. ( A ) nanovaccines containing OVA and α-Galcer or TLR-L were intravenously administered in mice. At different time points, mice were killed, sera were collected, and ELISA for IL-4 was performed. ( B ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At 6 or 24 h, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. Representative expression of pSTAT6 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( C and D ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. One day prior to vaccination, isotype or anti–IL-4 blocking antibodies were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and spleens from the 24-h time point were harvested. ( C ) Expression of CCR4 on OT-I T cells was evaluated by flow cytometry. ( D ) Expression of CCL17 in spleen cryosections (20 µm) was evaluated by confocal microscopy. (Scale bars, 50 µm.) ( E – H ) One-thousand CD8 + OT-I T cells were adoptively transferred prior to vaccination into CD45.1 mice. One day prior to vaccination, isotype, anti-CXCR3 ( E and F ), or anti-CCL17 or IL-4 antibodies ( G and H ) were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and 10 d later spleens were harvested and analyzed by flow cytometry. MPEC were defined as CD127 hi KLGR1 − cells and SLEC as CD127 lo KLGR1 + cells. Pool from 2 independent experiments with at least 5 mice per condition. Statistical analysis by 1-way ANOVA test for A and t test for C – H : * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Labeling, Staining, Flow Cytometry, Expressing, Blocking Assay, Injection, Confocal Microscopy

5) Product Images from "Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer"

Article Title: Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000203

IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P
Figure Legend Snippet: IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P

Techniques Used: Expressing, Transduction, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

5T4 expression on FFPE sections of tumor biopsies from patients with ovarian cancer. A, Light microscopy (×20 magnification) of FFPE sections after staining with a mouse anti-human 5T4 monoclonal antibody and hematoxylin. B, Percentage of EpCAM+ 5T4+ cells in the tumor disaggregates as determined by flow cytometry. C, Percentage of EpCAM positive tumor cells expressing 5T4. D, 5T4 expression on tumor samples as determined by H-score and mean fluorescence intensity (MFI). FFPE indicates formalin-fixed and paraffin-embedded.
Figure Legend Snippet: 5T4 expression on FFPE sections of tumor biopsies from patients with ovarian cancer. A, Light microscopy (×20 magnification) of FFPE sections after staining with a mouse anti-human 5T4 monoclonal antibody and hematoxylin. B, Percentage of EpCAM+ 5T4+ cells in the tumor disaggregates as determined by flow cytometry. C, Percentage of EpCAM positive tumor cells expressing 5T4. D, 5T4 expression on tumor samples as determined by H-score and mean fluorescence intensity (MFI). FFPE indicates formalin-fixed and paraffin-embedded.

Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded, Light Microscopy, Staining, Flow Cytometry, Cytometry, Fluorescence

Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P
Figure Legend Snippet: Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P

Techniques Used: Construct, Mouse Assay

5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P
Figure Legend Snippet: 5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P

Techniques Used: Construct, Transduction, Derivative Assay

Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.
Figure Legend Snippet: Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.

Techniques Used: Mouse Assay

6) Product Images from "Novel Identity and Functional Markers for Human Corneal Endothelial Cells"

Article Title: Novel Identity and Functional Markers for Human Corneal Endothelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.15-18826

Flow cytometry analysis of surface markers expression differentiates HCEC subpopulations in culture. ( A – C ) Three morphologically distinct HCEC cultures, canonical, mixed, and fibroblastic as marked, were carried forward for flow cytometry. ( D – G ) Surface marker expression by flow cytometry. Flow cytometry histograms are representative illustrations of each antibody expression profile. For quantification, a threshold at the top 1% of negative control cells was set to identify positive cells throughout all the independent experiments. Quantification of the percentage of positive cells for each marker showed that CD56 ( D ), CD248 ( E ), and CAR ( F ) expressions are low in a fibroblastic culture, while CD109 ( G ) is high. Data is representative of three or more independent experiments from separate corneal cultures (CD56: N = 10; CD109: N = 6; CD248: N = 3; CAR: N = 4; P values: #0.1; *
Figure Legend Snippet: Flow cytometry analysis of surface markers expression differentiates HCEC subpopulations in culture. ( A – C ) Three morphologically distinct HCEC cultures, canonical, mixed, and fibroblastic as marked, were carried forward for flow cytometry. ( D – G ) Surface marker expression by flow cytometry. Flow cytometry histograms are representative illustrations of each antibody expression profile. For quantification, a threshold at the top 1% of negative control cells was set to identify positive cells throughout all the independent experiments. Quantification of the percentage of positive cells for each marker showed that CD56 ( D ), CD248 ( E ), and CAR ( F ) expressions are low in a fibroblastic culture, while CD109 ( G ) is high. Data is representative of three or more independent experiments from separate corneal cultures (CD56: N = 10; CD109: N = 6; CD248: N = 3; CAR: N = 4; P values: #0.1; *

Techniques Used: Flow Cytometry, Cytometry, Expressing, Marker, Negative Control

( A – C ) Flow cytometry analysis by dual-color fluorescent dot plot histograms for the canonical ( A ), mixed ( B ), and fibroblastic ( C ) HCEC cultures show shift in the expression of CD56, CD248, CAR, and CD109 surface markers. Each graph is divided into four quadrants determined by the autofluorescence of unstained control cells as in Figure 4 , and gated to include 99% of unstained cells in the lower left quadrant (all negative markers). ( D – F ) Quantification of the percentage of canonical, fibroblastic, and mixed cell populations expressing markers tested in pairs, as marked. ( G ) Quantification of flow cytometry experiments showing no difference between canonical and fibroblastic cells in CD166/ALCAM, CD73, CD9, CD90, and β 1 Na + /K + ATPase expression. ( H ) Transendothelial electrical resistance assay using in vitro expanded HCECs whose CD56 expression had been determined by flow cytometry, demonstrating the greater ability of canonical CD56 high cells than fibroblastic CD56 low to form a barrier.
Figure Legend Snippet: ( A – C ) Flow cytometry analysis by dual-color fluorescent dot plot histograms for the canonical ( A ), mixed ( B ), and fibroblastic ( C ) HCEC cultures show shift in the expression of CD56, CD248, CAR, and CD109 surface markers. Each graph is divided into four quadrants determined by the autofluorescence of unstained control cells as in Figure 4 , and gated to include 99% of unstained cells in the lower left quadrant (all negative markers). ( D – F ) Quantification of the percentage of canonical, fibroblastic, and mixed cell populations expressing markers tested in pairs, as marked. ( G ) Quantification of flow cytometry experiments showing no difference between canonical and fibroblastic cells in CD166/ALCAM, CD73, CD9, CD90, and β 1 Na + /K + ATPase expression. ( H ) Transendothelial electrical resistance assay using in vitro expanded HCECs whose CD56 expression had been determined by flow cytometry, demonstrating the greater ability of canonical CD56 high cells than fibroblastic CD56 low to form a barrier.

Techniques Used: Flow Cytometry, Cytometry, Expressing, In Vitro

7) Product Images from "Distribution and Interaction of Murine Pulmonary Phagocytes in the Naive and Allergic Lung"

Article Title: Distribution and Interaction of Murine Pulmonary Phagocytes in the Naive and Allergic Lung

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01046

Localization of pulmonary dendritic cell (DC) and macrophage subsets by immunohistochemistry (IHC). Precision cut lung slices (PCLS) (300 µm) from naive C57BL/6 mice were generated and stained with anti-CD11c (green), anti-MHC-II or mPDCA-1 (turquois), anti-CD11b (purple), and anti-Langerin (red) ABs. Stained slices were evaluated with confocal microscopy. Conventional DCs (cDCs) and interstitial macrophages (IMs) were localized in the interstitium around AWs and pulmonary arteries (A) , around IAs (B) , and Vs (C) , while alveolar macrophages (AMs) were only located in the alveolar lumen (A–C) . Plasmacytoid DCs (pDCs) were primarily located in the alveolar interstitium (D) . Dashed lines indicate AWs and/or vessels. Abbreviations: AW, airway; PA, pulmonary artery; V, vein; IA, intra-acinar artery. Data are representative of at least three independent experiments.
Figure Legend Snippet: Localization of pulmonary dendritic cell (DC) and macrophage subsets by immunohistochemistry (IHC). Precision cut lung slices (PCLS) (300 µm) from naive C57BL/6 mice were generated and stained with anti-CD11c (green), anti-MHC-II or mPDCA-1 (turquois), anti-CD11b (purple), and anti-Langerin (red) ABs. Stained slices were evaluated with confocal microscopy. Conventional DCs (cDCs) and interstitial macrophages (IMs) were localized in the interstitium around AWs and pulmonary arteries (A) , around IAs (B) , and Vs (C) , while alveolar macrophages (AMs) were only located in the alveolar lumen (A–C) . Plasmacytoid DCs (pDCs) were primarily located in the alveolar interstitium (D) . Dashed lines indicate AWs and/or vessels. Abbreviations: AW, airway; PA, pulmonary artery; V, vein; IA, intra-acinar artery. Data are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Mouse Assay, Generated, Staining, Confocal Microscopy, Affinity Magnetic Separation, IA

Identification of lung phagocyte subsets by immunohistochemistry (IHC). Precision cut lung slices (PCLS) (300 µm) from naive C57BL/6 mice were generated and stained with anti-CD11c, anti-MHC-II or anti-mPDCA-1, anti-CD11b, and anti-Langerin (A,B) or anti-CD64 ABs (C,D) . Stained slices were evaluated with confocal microscopy. IHC of CD11b + dendritic cells (DCs), CD103 + DCs, and plasmacytoid DCs (pDCs) (A) or alveolar macrophages (AMs), interstitial macrophages (IM)1, and IM2 (C) . Single-color and merged color display with CD11c (green), MHC-II or mPDCA-1 (turquois), CD11b (purple), and Langerin (red) or CD64 (yellow). Data are representative of at least three independent experiments. Quantification of CD11b + DCs, CD103 + DCs, and pDCs (B) and AMs and IM2 (D) in total lungs. Lines indicate mean ± SEM. Differences between groups were tested by Kruskal–Wallis test (b) or Mann–Whitney U -test (d) for significance; * p
Figure Legend Snippet: Identification of lung phagocyte subsets by immunohistochemistry (IHC). Precision cut lung slices (PCLS) (300 µm) from naive C57BL/6 mice were generated and stained with anti-CD11c, anti-MHC-II or anti-mPDCA-1, anti-CD11b, and anti-Langerin (A,B) or anti-CD64 ABs (C,D) . Stained slices were evaluated with confocal microscopy. IHC of CD11b + dendritic cells (DCs), CD103 + DCs, and plasmacytoid DCs (pDCs) (A) or alveolar macrophages (AMs), interstitial macrophages (IM)1, and IM2 (C) . Single-color and merged color display with CD11c (green), MHC-II or mPDCA-1 (turquois), CD11b (purple), and Langerin (red) or CD64 (yellow). Data are representative of at least three independent experiments. Quantification of CD11b + DCs, CD103 + DCs, and pDCs (B) and AMs and IM2 (D) in total lungs. Lines indicate mean ± SEM. Differences between groups were tested by Kruskal–Wallis test (b) or Mann–Whitney U -test (d) for significance; * p

Techniques Used: Immunohistochemistry, Mouse Assay, Generated, Staining, Confocal Microscopy, Affinity Magnetic Separation, MANN-WHITNEY

8) Product Images from "Ephrinb1 and Ephrinb2 Are Associated with Interleukin-7 Receptor ? and Retard Its Internalization from the Cell Surface *"

Article Title: Ephrinb1 and Ephrinb2 Are Associated with Interleukin-7 Receptor ? and Retard Its Internalization from the Cell Surface *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.316414

IL-7Rα co-localizes with Efnb1 and Efnb2 after IL-7Rα/Efnb1 or IL-7Rα/Efnb2 cross-linking. Efnb1-EL4 cells ( A ) and Efnb2-EL4 cells ( B ) were cultured in plain medium (without cross-linking), or cross-linked with anti-Efnb1 plus
Figure Legend Snippet: IL-7Rα co-localizes with Efnb1 and Efnb2 after IL-7Rα/Efnb1 or IL-7Rα/Efnb2 cross-linking. Efnb1-EL4 cells ( A ) and Efnb2-EL4 cells ( B ) were cultured in plain medium (without cross-linking), or cross-linked with anti-Efnb1 plus

Techniques Used: Cell Culture

Efnb1/Efnb2 associate with IL-7Rα according to AB FRET. Efnb1-EL4 ( A ) and Efnb2-EL4 cells ( B ) were analyzed for interaction between Efnb1 and IL-7Rα, and between Efnb2 and IL-7Rα, respectively, by AB FRET. The cells were not Ab-cross-linked
Figure Legend Snippet: Efnb1/Efnb2 associate with IL-7Rα according to AB FRET. Efnb1-EL4 ( A ) and Efnb2-EL4 cells ( B ) were analyzed for interaction between Efnb1 and IL-7Rα, and between Efnb2 and IL-7Rα, respectively, by AB FRET. The cells were not Ab-cross-linked

Techniques Used:

Interaction between Efnb1/Efnb2 and IL-7Rα according to SE FRET and immunoprecipitation. A, SE FRET. Efnb1-EL4 cells and Efnb2-EL4 cells were cross-linked with a pair of Abs (anti-IL-7Rα or anti-Thy1.2 Ab plus anti-Efnb1 Ab for Efnb1-EL4
Figure Legend Snippet: Interaction between Efnb1/Efnb2 and IL-7Rα according to SE FRET and immunoprecipitation. A, SE FRET. Efnb1-EL4 cells and Efnb2-EL4 cells were cross-linked with a pair of Abs (anti-IL-7Rα or anti-Thy1.2 Ab plus anti-Efnb1 Ab for Efnb1-EL4

Techniques Used: Immunoprecipitation

9) Product Images from "Supramolecular PEGylation of biopharmaceuticals"

Article Title: Supramolecular PEGylation of biopharmaceuticals

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1616639113

Stability enhancement with other biopharmaceuticals. ( A ) Anti-CD20 antibody, aged in formulation with CB[7] or CB[7]–PEG, was evaluated for reactivity against CD20 + Raji cells, analyzed by flow cytometry after incubation with cells and subsequent
Figure Legend Snippet: Stability enhancement with other biopharmaceuticals. ( A ) Anti-CD20 antibody, aged in formulation with CB[7] or CB[7]–PEG, was evaluated for reactivity against CD20 + Raji cells, analyzed by flow cytometry after incubation with cells and subsequent

Techniques Used: Flow Cytometry, Cytometry, Incubation

10) Product Images from "β1-integrin–matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability"

Article Title: β1-integrin–matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1177/0271678X17722108

Changes in permeability (Papp) and tight junction (TJ) components of confluent primary cerebral microvessel endothelial cells at 24 h after β1-integrin–matrix interference. (a) Papp for 40 kDa-FITC-dextran significantly increased after Ha2/5 intervention compared to the vehicle control and isotype IgM (* p = 0.0029; n = 6 each). Papp under Ha2/5 was significantly higher than either control or isotype, which in turn did not differ ( α = 0.05 for all pairwise multiple comparisons). (b) Circumferential expression of TJ proteins at the inter-endothelial interface. Ha2/5 (white bars) simultaneously produced significant reductions in the circumferential expression (µm/cell) of the three TJ proteins by 24 h compared to the isotype IgM antibody (black bars) when the endothelium was grown on the different matrix substrates ( p
Figure Legend Snippet: Changes in permeability (Papp) and tight junction (TJ) components of confluent primary cerebral microvessel endothelial cells at 24 h after β1-integrin–matrix interference. (a) Papp for 40 kDa-FITC-dextran significantly increased after Ha2/5 intervention compared to the vehicle control and isotype IgM (* p = 0.0029; n = 6 each). Papp under Ha2/5 was significantly higher than either control or isotype, which in turn did not differ ( α = 0.05 for all pairwise multiple comparisons). (b) Circumferential expression of TJ proteins at the inter-endothelial interface. Ha2/5 (white bars) simultaneously produced significant reductions in the circumferential expression (µm/cell) of the three TJ proteins by 24 h compared to the isotype IgM antibody (black bars) when the endothelium was grown on the different matrix substrates ( p

Techniques Used: Permeability, Expressing, Produced

Alterations in cerebral microvessel endothelial cell β1-integrin expression in perfused cerebral tissues from adult murine β1-integrin conditional knockout preparations. (a) β1-integrin expression in two pairs each (left and right panels) of conditional knockout mice (β1 iKO ), after induction of the knockout with poly-I:C, compared with matched β1 +/+ litter-mate mice (that were Cre − β1-integrin flox/flox (β1 f/f) constructs, but induced with poly-I:C). The β1 +/+ mice displayed β1-integrin immunoreactivity homogeneously throughout all cerebral vessel diameters, while the β1 iKO mice demonstrated decreased microvessel β1-integrin expression through a range of microvessel diameters with regional heterogeneity. Left and right panels: β1-integrin immunoreactivity demonstrated 3-amino-9-ethylcarbazole (AEC) substrate (bluegray). Right panels: collagen IV immunoreactivity demonstrated with SG horse radish peroxidase substrate (red). Arrowheads indicate microvessels that display primarily collagen IV after conditional β1-integrin knockout in both sets. Note the greater intensity of the β1-integrin and the combined β1-integrin/collagen IV immunoreactivity in the respective β1 +/+ images. Magnification bars = 100 µm. (b) Two-color flow cytometry studies demonstrate two populations of CD31 + cerebral microvessel endothelial cells in β1 iKO subjects compared with a single population from the β1 +/+ litter-mates. Overall, the mean fluorescence intensity (MFI) for β1-integrin expression was significantly decreased in the β1 iKO endothelial cells compared to the β1 +/+ samples ( p = 0.05, single-tailed, n = 3). (c) Decreases in the microvessel basal lamina matrix content of collagen IV (see panel (a)) and laminin were observed in cerebral tissues from β1 iKO subjects (white bars) compared to β1 +/+ litter-mates (black bars) (** p
Figure Legend Snippet: Alterations in cerebral microvessel endothelial cell β1-integrin expression in perfused cerebral tissues from adult murine β1-integrin conditional knockout preparations. (a) β1-integrin expression in two pairs each (left and right panels) of conditional knockout mice (β1 iKO ), after induction of the knockout with poly-I:C, compared with matched β1 +/+ litter-mate mice (that were Cre − β1-integrin flox/flox (β1 f/f) constructs, but induced with poly-I:C). The β1 +/+ mice displayed β1-integrin immunoreactivity homogeneously throughout all cerebral vessel diameters, while the β1 iKO mice demonstrated decreased microvessel β1-integrin expression through a range of microvessel diameters with regional heterogeneity. Left and right panels: β1-integrin immunoreactivity demonstrated 3-amino-9-ethylcarbazole (AEC) substrate (bluegray). Right panels: collagen IV immunoreactivity demonstrated with SG horse radish peroxidase substrate (red). Arrowheads indicate microvessels that display primarily collagen IV after conditional β1-integrin knockout in both sets. Note the greater intensity of the β1-integrin and the combined β1-integrin/collagen IV immunoreactivity in the respective β1 +/+ images. Magnification bars = 100 µm. (b) Two-color flow cytometry studies demonstrate two populations of CD31 + cerebral microvessel endothelial cells in β1 iKO subjects compared with a single population from the β1 +/+ litter-mates. Overall, the mean fluorescence intensity (MFI) for β1-integrin expression was significantly decreased in the β1 iKO endothelial cells compared to the β1 +/+ samples ( p = 0.05, single-tailed, n = 3). (c) Decreases in the microvessel basal lamina matrix content of collagen IV (see panel (a)) and laminin were observed in cerebral tissues from β1 iKO subjects (white bars) compared to β1 +/+ litter-mates (black bars) (** p

Techniques Used: Expressing, Knock-Out, Mouse Assay, Construct, Flow Cytometry, Cytometry, Fluorescence

Gelatinase generation and secretion following β1-integrin–matrix interference in confluent primary cerebral endothelial cells. (a) Sample zymogram of (pro-)MMP-2 and (pro-)MMP-9 secreted from confluent primary murine cerebral microvascular endothelial cells exposed for 6 h to the interventions: vehicle control (lane 2), isotype IgM (lane 3), and Ha2/5 (lane 4), against human recombinant (pro-)MMP-2 (1 ng, lane 1a) and (pro-)MMP-9 (0.5 ng, lane 1b), which serve as markers. (b) Relative secretion of (pro-)MMP-2 following exposure to vehicle control (lane 2), isotype IgM (lane 3), and Ha2/5 (lane 4). No differences among these interventions were observed ( p = 0.96). Samples n = 6 separate cultures each. (c) Relative secretion of (pro-)MMP-9 following exposure to vehicle control (lane 2), isotype IgM (lane 3), and Ha2/5 (lane 4) from the same samples. No differences among these interventions were observed ( p = 0.09). Samples n = 6 separate cultures each. The three panels depict data from the same experiments. MMP: metalloproteinase.
Figure Legend Snippet: Gelatinase generation and secretion following β1-integrin–matrix interference in confluent primary cerebral endothelial cells. (a) Sample zymogram of (pro-)MMP-2 and (pro-)MMP-9 secreted from confluent primary murine cerebral microvascular endothelial cells exposed for 6 h to the interventions: vehicle control (lane 2), isotype IgM (lane 3), and Ha2/5 (lane 4), against human recombinant (pro-)MMP-2 (1 ng, lane 1a) and (pro-)MMP-9 (0.5 ng, lane 1b), which serve as markers. (b) Relative secretion of (pro-)MMP-2 following exposure to vehicle control (lane 2), isotype IgM (lane 3), and Ha2/5 (lane 4). No differences among these interventions were observed ( p = 0.96). Samples n = 6 separate cultures each. (c) Relative secretion of (pro-)MMP-9 following exposure to vehicle control (lane 2), isotype IgM (lane 3), and Ha2/5 (lane 4) from the same samples. No differences among these interventions were observed ( p = 0.09). Samples n = 6 separate cultures each. The three panels depict data from the same experiments. MMP: metalloproteinase.

Techniques Used: Recombinant

F-actin conformation changes following β1-integrin–matrix interference in confluent primary cerebral endothelial cells. (a) Left panel: fluorescent images of F-actin fibers (phalloidin) in confluent primary cerebral microvessel endothelial cells treated with vehicle control, isotype IgM, and β1-integrin-specific Ha2/5 antibodies from four separate animals (numbered) at 24 h after intervention. Montage of representative microscopic fields is from representative cultures derived from each animal subject. Right panel: montage of the binary images derived from the representative microscopic fields (fluorescent images of phalloidin) shown in the left panel. The binary images are generated as described in the “Materials and Methods” section and detailed in the Supplementary Materials. Magnification bar = 50 µm. (b) Time course of whole field phalloidin (F-actin) fluorescence intensity from cultures after intervention and conversion to binary data described in A above. The whole field fluorescence differed significantly by intervention (control, isotype IgM, or Ha2/5; p = 0.0002) and by time (3, 6, 18, or 24 h after intervention; p = 0.002). Each data point represents the mean of n = 4 observations. C: control; iso: isotype IgM. (c) Phalloidin (F-actin) fluorescence at 24 h differed significantly across the control, isotype IgM, and Ha2/5 interventions (* p
Figure Legend Snippet: F-actin conformation changes following β1-integrin–matrix interference in confluent primary cerebral endothelial cells. (a) Left panel: fluorescent images of F-actin fibers (phalloidin) in confluent primary cerebral microvessel endothelial cells treated with vehicle control, isotype IgM, and β1-integrin-specific Ha2/5 antibodies from four separate animals (numbered) at 24 h after intervention. Montage of representative microscopic fields is from representative cultures derived from each animal subject. Right panel: montage of the binary images derived from the representative microscopic fields (fluorescent images of phalloidin) shown in the left panel. The binary images are generated as described in the “Materials and Methods” section and detailed in the Supplementary Materials. Magnification bar = 50 µm. (b) Time course of whole field phalloidin (F-actin) fluorescence intensity from cultures after intervention and conversion to binary data described in A above. The whole field fluorescence differed significantly by intervention (control, isotype IgM, or Ha2/5; p = 0.0002) and by time (3, 6, 18, or 24 h after intervention; p = 0.002). Each data point represents the mean of n = 4 observations. C: control; iso: isotype IgM. (c) Phalloidin (F-actin) fluorescence at 24 h differed significantly across the control, isotype IgM, and Ha2/5 interventions (* p

Techniques Used: Derivative Assay, Generated, Fluorescence

F-actin and claudin-5 expression following β1-integrin–matrix interference in the same primary cerebral endothelial cells from confluent cultures using three-color flow cytometry. (a) Sample distributions of β1-integrin, F-actin, and claudin-5 expression in confluent primary cerebral endothelial cells (from the top) that were subject to isotype IgM (blue) or interference with β1-integrin–collagen IV adhesion by Ha2/5 (red). Note the bimodal distribution of each epitope, and the loss of β1-integrin (quantified in panel C) and changes in claudin-5 in the high expression subpopulation (seen in panel B, bottom pair). (b) Changes in the distributions of endothelial cell β1-integrin, F-actin, and claudin-5 shown in panel A from isotype IgM to interference in β1-integrin–collagen IV adhesion by Ha2/5. Note the altered distribution of claudin-5 with loss of β1-integrin, but the relatively unchanged distribution of F-actin vs. claudin-5 (middle pair). (c) The mean fluorescence intensities (MFI) of β1-integrin, F-actin, and claudin-5 described following treatment with isotype IgM (black bars) or Ha2/5 (white bars) antibodies. The MFIs differed significantly by intervention ( p
Figure Legend Snippet: F-actin and claudin-5 expression following β1-integrin–matrix interference in the same primary cerebral endothelial cells from confluent cultures using three-color flow cytometry. (a) Sample distributions of β1-integrin, F-actin, and claudin-5 expression in confluent primary cerebral endothelial cells (from the top) that were subject to isotype IgM (blue) or interference with β1-integrin–collagen IV adhesion by Ha2/5 (red). Note the bimodal distribution of each epitope, and the loss of β1-integrin (quantified in panel C) and changes in claudin-5 in the high expression subpopulation (seen in panel B, bottom pair). (b) Changes in the distributions of endothelial cell β1-integrin, F-actin, and claudin-5 shown in panel A from isotype IgM to interference in β1-integrin–collagen IV adhesion by Ha2/5. Note the altered distribution of claudin-5 with loss of β1-integrin, but the relatively unchanged distribution of F-actin vs. claudin-5 (middle pair). (c) The mean fluorescence intensities (MFI) of β1-integrin, F-actin, and claudin-5 described following treatment with isotype IgM (black bars) or Ha2/5 (white bars) antibodies. The MFIs differed significantly by intervention ( p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence

Inhibition of established endothelial cell β1-integrin–collagen IV interactions induce MLC phosphorylation. (a) Effect of Rho kinase (ROCK) and MLC kinase (MLCK) inhibitors on endothelial cell pMLC and MLC expression during Ha2/5 exposure. Logged optical density ratios of pMLC/β-actin, MLC/β-actin, and pMLC/MLC differed significantly across the four conditions isotype IgM, Ha2/5, Ha2/5 + Y-27632, and Ha2/5 + IP-I8 ( F 3,20 = 5.18, p = 0.0083; F 3,20 = 4.59, p = 0.0133; and F 3,20 = 4.61, p = 0.0132, respectively). The ROCK inhibitor Y-27632 had a significant effect on the pMLC/MLC ratios generated by Ha2/5 as demonstrated by a post hoc Bonferroni comparison of the logged optical density ratios (* at α = 0.05). Immunoblot examples are shown in insets. pM: phosphoMLC; M: MLC; arrow: β-actin. Samples were taken from n = 6 separate cultures each. (b) The increased permeability of the endothelial cell monolayers to 40 kDa-dextran at 6 h caused by Ha2/5 was abrogated when inhibitor Y-27632 and IP-I8 were together with Ha2/5. From a post hoc Bonferroni comparison of the Papp by condition, both isotype IgM (black bar) and isotype IgM + Y-27632 + IP-I8 differed significantly from both Ha2/5 (white bar) and Ha2/5 + Y-27632. Ha2/5 differed significantly from Ha2/5 + Y-27632 + IP-I8 (at α = 0.05). Samples were taken from n = 14 (isotype IgM), 6 (isotype IgM + Y-27632 + IP-I8), 14 (Ha2/5), 8 (Ha2/5 + Y-27632), 8 (Ha2/5 + IP-I8), and 6 (Ha2/5 + Y-27632 + IP-I8) cultures. There was no effect on Papp at 24 h ( p = 0.9012). (c) ILK suppression altered endothelial cell β1-integrin, F-actin, and claudin-5 expression (MFI of each, in three-color flow cytometry) by 24 h after transfection. From a general linear model analysis, the MFIs differed significantly by intervention (control (shaded bars), control-siRNA (black bars), or ILK-siRNA (white bars): F 2,63 = 20.12, p
Figure Legend Snippet: Inhibition of established endothelial cell β1-integrin–collagen IV interactions induce MLC phosphorylation. (a) Effect of Rho kinase (ROCK) and MLC kinase (MLCK) inhibitors on endothelial cell pMLC and MLC expression during Ha2/5 exposure. Logged optical density ratios of pMLC/β-actin, MLC/β-actin, and pMLC/MLC differed significantly across the four conditions isotype IgM, Ha2/5, Ha2/5 + Y-27632, and Ha2/5 + IP-I8 ( F 3,20 = 5.18, p = 0.0083; F 3,20 = 4.59, p = 0.0133; and F 3,20 = 4.61, p = 0.0132, respectively). The ROCK inhibitor Y-27632 had a significant effect on the pMLC/MLC ratios generated by Ha2/5 as demonstrated by a post hoc Bonferroni comparison of the logged optical density ratios (* at α = 0.05). Immunoblot examples are shown in insets. pM: phosphoMLC; M: MLC; arrow: β-actin. Samples were taken from n = 6 separate cultures each. (b) The increased permeability of the endothelial cell monolayers to 40 kDa-dextran at 6 h caused by Ha2/5 was abrogated when inhibitor Y-27632 and IP-I8 were together with Ha2/5. From a post hoc Bonferroni comparison of the Papp by condition, both isotype IgM (black bar) and isotype IgM + Y-27632 + IP-I8 differed significantly from both Ha2/5 (white bar) and Ha2/5 + Y-27632. Ha2/5 differed significantly from Ha2/5 + Y-27632 + IP-I8 (at α = 0.05). Samples were taken from n = 14 (isotype IgM), 6 (isotype IgM + Y-27632 + IP-I8), 14 (Ha2/5), 8 (Ha2/5 + Y-27632), 8 (Ha2/5 + IP-I8), and 6 (Ha2/5 + Y-27632 + IP-I8) cultures. There was no effect on Papp at 24 h ( p = 0.9012). (c) ILK suppression altered endothelial cell β1-integrin, F-actin, and claudin-5 expression (MFI of each, in three-color flow cytometry) by 24 h after transfection. From a general linear model analysis, the MFIs differed significantly by intervention (control (shaded bars), control-siRNA (black bars), or ILK-siRNA (white bars): F 2,63 = 20.12, p

Techniques Used: Inhibition, Expressing, Generated, Permeability, Flow Cytometry, Cytometry, Transfection

11) Product Images from "The CD300e molecule in mice is an immune-activating receptor"

Article Title: The CD300e molecule in mice is an immune-activating receptor

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA117.000696

mCD300e is highly expressed in CD115 + Ly-6C low/int monocytes. A, mouse PB cells were stained with FITC-conjugated anti-CD3, CD19, or Ly-6G Ab and with biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. Expression of mCD300e in CD3 + , CD19 + , or Ly-6G + cells was shown. B, mouse PB cells were stained with FITC-conjugated CD11b, CD11c, F4/80, Ly-6C, CD80, CD86, or MHC class II (MHC-II) Ab and with biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. C, mouse PB cells were stained with FITC-conjugated anti-Ly-6C Ab and PE-conjugated anti-CD115 Ab ( upper left panel ) or FITC-conjugated anti-Ly-6C Ab and biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin ( lower left panel ). Histograms showed expression of Ly-6C in CD115 + PB cells ( upper right panel ) or in mCD300e + PB cells ( lower right panel ). D, mean fluorescent intensity ( MFI ) of mCD300e in CD115 + Ly-6C low , CD115 + Ly-6C int , or CD115 + Ly-6C high PB monocytes was measured by flow cytometry. E, BM, spleen, or thymus cells were stained with FITC-conjugated anti-CD80 Ab and biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. F, BMMCs, BMmDCs, BMpDCs, or BMMφ were stained with biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. G, MFI of mCD300e in CD11c + PB monocytes from WT, FcR γ −/− , DAP12 −/− , or FcR γ −/− DAP12 −/− mice were measured by flow cytometry. A–C, E, and F, data are representative of three independent experiments. D and G , all data points correspond to the mean ± S.D. of four independent experiments. Statistically significant differences are shown. *, p
Figure Legend Snippet: mCD300e is highly expressed in CD115 + Ly-6C low/int monocytes. A, mouse PB cells were stained with FITC-conjugated anti-CD3, CD19, or Ly-6G Ab and with biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. Expression of mCD300e in CD3 + , CD19 + , or Ly-6G + cells was shown. B, mouse PB cells were stained with FITC-conjugated CD11b, CD11c, F4/80, Ly-6C, CD80, CD86, or MHC class II (MHC-II) Ab and with biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. C, mouse PB cells were stained with FITC-conjugated anti-Ly-6C Ab and PE-conjugated anti-CD115 Ab ( upper left panel ) or FITC-conjugated anti-Ly-6C Ab and biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin ( lower left panel ). Histograms showed expression of Ly-6C in CD115 + PB cells ( upper right panel ) or in mCD300e + PB cells ( lower right panel ). D, mean fluorescent intensity ( MFI ) of mCD300e in CD115 + Ly-6C low , CD115 + Ly-6C int , or CD115 + Ly-6C high PB monocytes was measured by flow cytometry. E, BM, spleen, or thymus cells were stained with FITC-conjugated anti-CD80 Ab and biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. F, BMMCs, BMmDCs, BMpDCs, or BMMφ were stained with biotinylated anti-mCD300e Ab or Armenian hamster IgG Ab followed by PE-conjugated streptavidin. G, MFI of mCD300e in CD11c + PB monocytes from WT, FcR γ −/− , DAP12 −/− , or FcR γ −/− DAP12 −/− mice were measured by flow cytometry. A–C, E, and F, data are representative of three independent experiments. D and G , all data points correspond to the mean ± S.D. of four independent experiments. Statistically significant differences are shown. *, p

Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Mouse Assay

12) Product Images from "The Expression of BAFF, APRIL and TWEAK Is Altered in Eczema Skin but Not in the Circulation of Atopic and Seborrheic Eczema Patients"

Article Title: The Expression of BAFF, APRIL and TWEAK Is Altered in Eczema Skin but Not in the Circulation of Atopic and Seborrheic Eczema Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022202

Expression of BAFF in skin of HC and lesional skin of AE and SE determined by quantitative RT-PCR and immunofluorescence. (A–B) Relative mRNA expression of BAFF, BAFFR and TACI in skin of HC (n = 9), lesional skin of AE (n = 9), APT-AE (n = 6) and SE (n = 6). Data shown as mean ± SEM, *p
Figure Legend Snippet: Expression of BAFF in skin of HC and lesional skin of AE and SE determined by quantitative RT-PCR and immunofluorescence. (A–B) Relative mRNA expression of BAFF, BAFFR and TACI in skin of HC (n = 9), lesional skin of AE (n = 9), APT-AE (n = 6) and SE (n = 6). Data shown as mean ± SEM, *p

Techniques Used: Expressing, Quantitative RT-PCR, Immunofluorescence

13) Product Images from "The actin-bundling protein L-plastin is essential for marginal zone B cell development"

Article Title: The actin-bundling protein L-plastin is essential for marginal zone B cell development

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1101033

Increased numbers of lymphocytes in the blood of LPL −/− mice. (A) Total cell number and number of B220 + cells isolated from blood of WT and LPL −/− mice. (B) Flow cytometric analysis of IgM and IgD expression on peripheral
Figure Legend Snippet: Increased numbers of lymphocytes in the blood of LPL −/− mice. (A) Total cell number and number of B220 + cells isolated from blood of WT and LPL −/− mice. (B) Flow cytometric analysis of IgM and IgD expression on peripheral

Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Expressing

14) Product Images from "The Stalk Domain of NKp30 Contributes to Ligand Binding and Signaling of a Preassembled NKp30-CD3ζ Complex *"

Article Title: The Stalk Domain of NKp30 Contributes to Ligand Binding and Signaling of a Preassembled NKp30-CD3ζ Complex *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.742981

Positioning of amino acids at the membrane transition interface. A , schematic representation of the N -glycosylation scanning. Amino acids mutated to N -glycosylation acceptor sites are shown in bold , and amino acids to be analyzed at the membrane transition interface are shown in red. Brackets indicate the 14 amino acids between N -glycosylation sites and the amino acids to be analyzed. Solid circles , glycosylated sites after mutation; open circles , non-glycosylated sites after mutation. B and C , plasma membrane and intracellular expression of the NKp30 ( B ) and NKp46 ( C ) mutants in transduced 293T/17 cells analyzed by flow cytometry. D and E , Western blotting analysis of NKp30 ( D ) and NKp46 ( E ) mutants detected with NKp30- or NKp46-specific antibodies, respectively. One representative experiment of three is shown.
Figure Legend Snippet: Positioning of amino acids at the membrane transition interface. A , schematic representation of the N -glycosylation scanning. Amino acids mutated to N -glycosylation acceptor sites are shown in bold , and amino acids to be analyzed at the membrane transition interface are shown in red. Brackets indicate the 14 amino acids between N -glycosylation sites and the amino acids to be analyzed. Solid circles , glycosylated sites after mutation; open circles , non-glycosylated sites after mutation. B and C , plasma membrane and intracellular expression of the NKp30 ( B ) and NKp46 ( C ) mutants in transduced 293T/17 cells analyzed by flow cytometry. D and E , Western blotting analysis of NKp30 ( D ) and NKp46 ( E ) mutants detected with NKp30- or NKp46-specific antibodies, respectively. One representative experiment of three is shown.

Techniques Used: Mutagenesis, Expressing, Flow Cytometry, Cytometry, Western Blot

Contribution of individual amino acids of the NKp30 stalk to signaling. A–C , A5-GFP cells transduced with the different NKp30 alanine mutants or a lysine mutant of Arg-143 were analyzed for their signaling capacity after co-incubation with Ba/F3 B7-H6 cells ( A and C ) or after stimulation with plate-bound NKp30-specific antibodies ( B ). GFP expression was analyzed by flow cytometry of CD4 + /SytoxBlue − A5-GFP cells. The percentage of GFP-positive cells normalized to wild type is indicated as the mean ± S.E. of three independent experiments measured in duplicate. Statistical significance of flow cytometry experiments was assessed by one-way ANOVA and Dunnett's multiple comparisons test with Prism 6 software. *, p = 0.01–0.05; **, p = 0.001–0.01; ***, p = 0.0001–0.001; ****, p
Figure Legend Snippet: Contribution of individual amino acids of the NKp30 stalk to signaling. A–C , A5-GFP cells transduced with the different NKp30 alanine mutants or a lysine mutant of Arg-143 were analyzed for their signaling capacity after co-incubation with Ba/F3 B7-H6 cells ( A and C ) or after stimulation with plate-bound NKp30-specific antibodies ( B ). GFP expression was analyzed by flow cytometry of CD4 + /SytoxBlue − A5-GFP cells. The percentage of GFP-positive cells normalized to wild type is indicated as the mean ± S.E. of three independent experiments measured in duplicate. Statistical significance of flow cytometry experiments was assessed by one-way ANOVA and Dunnett's multiple comparisons test with Prism 6 software. *, p = 0.01–0.05; **, p = 0.001–0.01; ***, p = 0.0001–0.001; ****, p

Techniques Used: Transduction, Mutagenesis, Incubation, Expressing, Flow Cytometry, Cytometry, Software

Folding, plasma membrane targeting, and retention of NKp30 and NKp46 depend on CD3ζ. A , immunofluorescence staining of CD3ζ-transduced HeLa cells additionally transduced with either NKp30 or NKp46. Red , surface staining of the NCRs with specific antibodies; green , intracellular staining of CD3ζ with specific antibodies; blue , DAPI; size bar , 10 μm. One representative picture of at least five is shown. B , immunofluorescence microscopy analysis of NKp30 and NKp46 expression (plasma membrane and intracellular) in HeLa cells with or without CD3ζ. Red , staining of the NCRs with specific antibodies; size bar , 10 μm. One representative picture of at least five is shown. C , flow cytometric analysis of NKp30 and NKp46 expression (plasma membrane and intracellular) in HeLa cells with or without CD3ζ. Gray , isotype control; red , anti-NKp30; blue , anti-NKp46. Median fluorescence intensity ( MFI ) ratios (MFI of NCR staining normalized to MFI of isotype control staining) are indicated. One representative experiment of three is shown.
Figure Legend Snippet: Folding, plasma membrane targeting, and retention of NKp30 and NKp46 depend on CD3ζ. A , immunofluorescence staining of CD3ζ-transduced HeLa cells additionally transduced with either NKp30 or NKp46. Red , surface staining of the NCRs with specific antibodies; green , intracellular staining of CD3ζ with specific antibodies; blue , DAPI; size bar , 10 μm. One representative picture of at least five is shown. B , immunofluorescence microscopy analysis of NKp30 and NKp46 expression (plasma membrane and intracellular) in HeLa cells with or without CD3ζ. Red , staining of the NCRs with specific antibodies; size bar , 10 μm. One representative picture of at least five is shown. C , flow cytometric analysis of NKp30 and NKp46 expression (plasma membrane and intracellular) in HeLa cells with or without CD3ζ. Gray , isotype control; red , anti-NKp30; blue , anti-NKp46. Median fluorescence intensity ( MFI ) ratios (MFI of NCR staining normalized to MFI of isotype control staining) are indicated. One representative experiment of three is shown.

Techniques Used: Immunofluorescence, Staining, Transduction, Microscopy, Expressing, Flow Cytometry, Fluorescence

15) Product Images from "PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells"

Article Title: PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells

Journal: Molecular Systems Biology

doi: 10.1038/msb.2012.56

PLAU is highly expressed in Tregs. ( A ) mRNA expression of PLAU assessed by HTR time-series microarray in the first 6 h following anti-CD28/-CD3/IL2 stimulation. ( B ) Single staining of surface expression of PLAU assessed by FACS for the first day. For the first 6 h, the cells were restimulated by anti-CD28/-CD3/IL2, but for the rest of the sampling time, the cells were independently restimulated by EBV-B cells daily treated with IL2. ‘Max', maximum. ( C ) Single staining of surface expression of PLAU assessed by FACS at time points from day 3 to day 12. ( D ) Coexpression of PLAU and FOXP3 assessed by FACS on day 3 and day 7 following EBV-B cell restimulation. Enlarged red numbers indicate the overall percentage of the two right quadrants (i.e., FOXP3 + cells). Data represent three independent experiments.
Figure Legend Snippet: PLAU is highly expressed in Tregs. ( A ) mRNA expression of PLAU assessed by HTR time-series microarray in the first 6 h following anti-CD28/-CD3/IL2 stimulation. ( B ) Single staining of surface expression of PLAU assessed by FACS for the first day. For the first 6 h, the cells were restimulated by anti-CD28/-CD3/IL2, but for the rest of the sampling time, the cells were independently restimulated by EBV-B cells daily treated with IL2. ‘Max', maximum. ( C ) Single staining of surface expression of PLAU assessed by FACS at time points from day 3 to day 12. ( D ) Coexpression of PLAU and FOXP3 assessed by FACS on day 3 and day 7 following EBV-B cell restimulation. Enlarged red numbers indicate the overall percentage of the two right quadrants (i.e., FOXP3 + cells). Data represent three independent experiments.

Techniques Used: Expressing, Microarray, Staining, FACS, Sampling

Low expression of effector cytokines, high expression of CCL20 , FOXP3, and GARP ( LRRC32 ) in stimulated Tregs versus Teffs in the first 6 h and GARP subnetwork. ( A ) The kinetics of transcriptional expression of chosen cytokines in the first 6 h assessed by HTR time-series data. Treg1 and Treg2 (or Teff1 and Teff2) are the two independent repeated HTR time-series experiments for Tregs (or Teffs). Numbers on the x -axis indicate the corresponding sampling time points. Time point 1 is the activation starting time zero and each interval is 20 min. ( B ) The kinetics of CCL20 (left), FOXP3 (middle), and GARP (right) expression in the first 6 h assessed by HTR time-series data. ( C ) The GARP subnetwork extracted from the constructed Treg-specific correlation network based on the HTR transcription data by the proposed strategy. Each oval represents one gene. Each line between GARP and the other genes represents a correlation linkage.
Figure Legend Snippet: Low expression of effector cytokines, high expression of CCL20 , FOXP3, and GARP ( LRRC32 ) in stimulated Tregs versus Teffs in the first 6 h and GARP subnetwork. ( A ) The kinetics of transcriptional expression of chosen cytokines in the first 6 h assessed by HTR time-series data. Treg1 and Treg2 (or Teff1 and Teff2) are the two independent repeated HTR time-series experiments for Tregs (or Teffs). Numbers on the x -axis indicate the corresponding sampling time points. Time point 1 is the activation starting time zero and each interval is 20 min. ( B ) The kinetics of CCL20 (left), FOXP3 (middle), and GARP (right) expression in the first 6 h assessed by HTR time-series data. ( C ) The GARP subnetwork extracted from the constructed Treg-specific correlation network based on the HTR transcription data by the proposed strategy. Each oval represents one gene. Each line between GARP and the other genes represents a correlation linkage.

Techniques Used: Expressing, Sampling, Activation Assay, Construct

PLAU high Tregs exhibit higher potential in suppressing effector T cells and express higher levels of Treg-important genes, but they express lower levels of cytokine genes compared to PLAU low Tregs. ( A ) PLAU high and PLAU low Tregs were sorted by PLAU expression level (left, isotype control; the figure in the right column is divided into three parts: negative, low, and high levels of PLAU expression). ( B ) FACS of the PLAU high (red line) and PLAU low Tregs (black line) immediately after sorting. ( C ) The suppression assay of different cells populations assessed by uptake of [H 3 ]thymidine (H3-TdR) (refer to Materials and methods). Error bars represent s.d. values. Results are representative of three independent experiments. ( D ) Higher expression of PLAU, FOXP3, and GARP in PLAU high than PLAU low Tregs assessed by FACS at day 3 post restimulation. The PLAU subsets of Tregs were expanded for at least four rounds. Of note, in this panel EBV-B cells were not sorted out before FACS analysis. Results are representative of three independent experiments. ( E ) The CFSE dilution measurement of the Teff proliferation co-cultured with Tregs at various ratios of PLAU_high (upper row) or PLAU_low (lower row) Tregs to Teffs as well as co-cultured EBV-B cells after 4 days. Number in each histogram represents the percentage of dividing cells from the total population (the total population indicates the gated DAPI-negative and CD4 + , CFSE stained Teffs). Results are representative of three independent experiments. ( F ) Higher mRNA levels of FOXP3 , GARP , LGMN , CTLA4 , and EOS in PLAU high Tregs assessed by real-time qPCR analysis. The numbers on the y axis indicate fold changes relative to RPS9 . The numbers under each subfigure represent the increased fold changes in PLAU high Tregs compared to PLAU low Tregs. The qPCR analysis was performed after the removal of B cells by FACS before extracting RNA. The P -values represented by ‘*' symbols indicate the results from a one-tailed Student's t -test for three replicates. Results are representative of three independent experiments. ( G) PLAU low Tregs reversed the expression of Treg-repressed cytokines as assessed by a human Th1-Th2-Th3 cytokine PCR array. Left, numbers on the y axis indicate fold changes relative to actin. Data represent two independent experiments. The reported genes are the overlap of the two independent experiments, which show at least 1.5-fold decrease in PLAU high versus PLAU low Tregs. Right, Venn diagram of the two independent experiments. The numbers specify the number of genes that decreased at least 1.5-fold. ( H ) Independent real-time PCR confirmed the expression of the Treg-repressed cytokines in (G), such as IL5 , IL13 , and CSF2 . The percentage under each subfigure represents the decreased percentage of the corresponding gene in PLAU high versus PLAU low Tregs. Error bars represent s.d. values. The P -values represented by ‘*' symbols indicate the results from a one-tailed Student's t -test for three replicates.
Figure Legend Snippet: PLAU high Tregs exhibit higher potential in suppressing effector T cells and express higher levels of Treg-important genes, but they express lower levels of cytokine genes compared to PLAU low Tregs. ( A ) PLAU high and PLAU low Tregs were sorted by PLAU expression level (left, isotype control; the figure in the right column is divided into three parts: negative, low, and high levels of PLAU expression). ( B ) FACS of the PLAU high (red line) and PLAU low Tregs (black line) immediately after sorting. ( C ) The suppression assay of different cells populations assessed by uptake of [H 3 ]thymidine (H3-TdR) (refer to Materials and methods). Error bars represent s.d. values. Results are representative of three independent experiments. ( D ) Higher expression of PLAU, FOXP3, and GARP in PLAU high than PLAU low Tregs assessed by FACS at day 3 post restimulation. The PLAU subsets of Tregs were expanded for at least four rounds. Of note, in this panel EBV-B cells were not sorted out before FACS analysis. Results are representative of three independent experiments. ( E ) The CFSE dilution measurement of the Teff proliferation co-cultured with Tregs at various ratios of PLAU_high (upper row) or PLAU_low (lower row) Tregs to Teffs as well as co-cultured EBV-B cells after 4 days. Number in each histogram represents the percentage of dividing cells from the total population (the total population indicates the gated DAPI-negative and CD4 + , CFSE stained Teffs). Results are representative of three independent experiments. ( F ) Higher mRNA levels of FOXP3 , GARP , LGMN , CTLA4 , and EOS in PLAU high Tregs assessed by real-time qPCR analysis. The numbers on the y axis indicate fold changes relative to RPS9 . The numbers under each subfigure represent the increased fold changes in PLAU high Tregs compared to PLAU low Tregs. The qPCR analysis was performed after the removal of B cells by FACS before extracting RNA. The P -values represented by ‘*' symbols indicate the results from a one-tailed Student's t -test for three replicates. Results are representative of three independent experiments. ( G) PLAU low Tregs reversed the expression of Treg-repressed cytokines as assessed by a human Th1-Th2-Th3 cytokine PCR array. Left, numbers on the y axis indicate fold changes relative to actin. Data represent two independent experiments. The reported genes are the overlap of the two independent experiments, which show at least 1.5-fold decrease in PLAU high versus PLAU low Tregs. Right, Venn diagram of the two independent experiments. The numbers specify the number of genes that decreased at least 1.5-fold. ( H ) Independent real-time PCR confirmed the expression of the Treg-repressed cytokines in (G), such as IL5 , IL13 , and CSF2 . The percentage under each subfigure represents the decreased percentage of the corresponding gene in PLAU high versus PLAU low Tregs. Error bars represent s.d. values. The P -values represented by ‘*' symbols indicate the results from a one-tailed Student's t -test for three replicates.

Techniques Used: Expressing, FACS, Suppression Assay, Cell Culture, Staining, Real-time Polymerase Chain Reaction, One-tailed Test, Polymerase Chain Reaction

Knockdown of PLAU downregulates FOXP3 and other known important Treg genes. ( A ) Knockdown of PLAU by electroporation-mediated siRNA assessed by FACS. Before performing the knockdown experiments, we first restimulated the Tregs by co-culturing them with EBV-B cells for 3 days. To remove the B cells, we collected CD4 + cells by FACS before the knockdown experiments. The Tregs were rested for another 3 days after siRNA transfection and were then analyzed by FACS. ( B ) Left, the gated Tregs, PLAU expression assessed by FACS; right, the gated Tregs, expression of FOXP3 protein assessed by FACS. Enlarged numbers indicate the geometric mean of fluorescence intensity of the corresponding protein. ‘Max', maximum. ( C ) Downregulation of mRNA levels of PLAU , FOXP3 , EOS , CTLA4 , and LGMN assessed by real-time qPCR analysis. The numbers on the y axis indicate fold change relative to RPS9 ( Bruder et al, 2004 ). The percentage under each subfigure represents the decreased percentage of the corresponding gene in Tregs transfected with specific siRNA against PLAU (si_PLAU) compared to Tregs transfected with non-silencing siRNA (si_NS). Error bars represent standard deviation (s.d.) values. The P -values indicate the results from a one-tailed Student's t -test for three repeated measurements. P ≤0.05 is indicated by * P ≤0.01, by ** P ≤0.001, and by *** for all the figures. The qPCR results displayed here were from experiments performed on day 3 post siRNA transfection for all the genes, except for LGMN on day 2 post siRNA. Data represent two to four independent experiments.
Figure Legend Snippet: Knockdown of PLAU downregulates FOXP3 and other known important Treg genes. ( A ) Knockdown of PLAU by electroporation-mediated siRNA assessed by FACS. Before performing the knockdown experiments, we first restimulated the Tregs by co-culturing them with EBV-B cells for 3 days. To remove the B cells, we collected CD4 + cells by FACS before the knockdown experiments. The Tregs were rested for another 3 days after siRNA transfection and were then analyzed by FACS. ( B ) Left, the gated Tregs, PLAU expression assessed by FACS; right, the gated Tregs, expression of FOXP3 protein assessed by FACS. Enlarged numbers indicate the geometric mean of fluorescence intensity of the corresponding protein. ‘Max', maximum. ( C ) Downregulation of mRNA levels of PLAU , FOXP3 , EOS , CTLA4 , and LGMN assessed by real-time qPCR analysis. The numbers on the y axis indicate fold change relative to RPS9 ( Bruder et al, 2004 ). The percentage under each subfigure represents the decreased percentage of the corresponding gene in Tregs transfected with specific siRNA against PLAU (si_PLAU) compared to Tregs transfected with non-silencing siRNA (si_NS). Error bars represent standard deviation (s.d.) values. The P -values indicate the results from a one-tailed Student's t -test for three repeated measurements. P ≤0.05 is indicated by * P ≤0.01, by ** P ≤0.001, and by *** for all the figures. The qPCR results displayed here were from experiments performed on day 3 post siRNA transfection for all the genes, except for LGMN on day 2 post siRNA. Data represent two to four independent experiments.

Techniques Used: Electroporation, FACS, Transfection, Expressing, Fluorescence, Real-time Polymerase Chain Reaction, Standard Deviation, One-tailed Test

Elevated thymic Foxp3 + Treg population while diminished suppressive capability of Cd44 high Cd66L low Tregs in Plau -KO mice. ( A ) Percentages of Cd4 + SP cells among murine thymocytes isolated from wild-type (WT) control mice and Plau heterozygous knockout mice ( Plau +/ − ) assessed by FACS. ( B ) Percentages of Cd25 + Foxp3 + cells among murine Cd4 + SP thymocytes isolated from WT and Plau +/ − mice assessed by FACS. Only gated Cd4 + SP cells from (A) are shown in (B). Data represent one of the four mice per group. ( C ) Comparison of percentages of Cd25 + Foxp3 + cells among murine Cd4 + SP thymocytes between 3-week-old WT and Plau +/ − groups. Data are representative of two independent experiments. ( D ) Left, comparison of percentages of Cd25 + Foxp3 + cells among murine total Cd4 + thymocytes between WT and Plau +/ − and/or Plau −/ − groups, aged 12, 10 and 3 weeks. Error bars represent s.d. values. P -value represented by ‘*' symbols indicate results from a one-tailed Student's t -test for n mice per group (the corresponding number n per group is indicated under each group column). Right, comparison of percentages of Cd25 + Foxp3 + cells among murine total Cd4 + splenocytes between WT, Plau +/ − and Plau −/− groups in 10-week-old mice. ( E ) Comparison of suppressive capability of Cd4 + Cd25 + Tregs between littermate WT, Plau +/ − , and Plau −/− groups, age ≥20 weeks. Left, proliferation assessed by uptake of [H3]thymidine. The P -value above each ratio represented by ‘*' symbols indicates the results from a one-tailed Student's t -test between WT and Plau −/− groups. Right, proliferation assessed by the CFSE dilution measurement of Cd25 − Ths co-cultured with Plau −/− or WT Tregs as well as irradiated feeder cells and Cd3 at various ratios of Tregs to Teffs after 3.5 days. Number in each histogram represents percentage of dividing cells from the total population (the total population indicates the gated DAPI-negative and CD4 + , CFSE stained Ths). ( F ) Left, the gating scheme used to analyze frequencies of Cd44 high Cd62L low Tregs. Right, comparison of percentages of Cd44 high Cd62L low Tregs among total Tregs in spleen (SPL), peripheral LN (pLN) or mesenteric LN (mLN) from WT and Plau +/ − and/or Plau −/ − between groups of mice aged 24 and 3 weeks. The P -value represented by ‘*' symbols indicates the results from a one-tailed Student's t -test. ( G ) Cd44 high Cd62L low Tregs from Plau –/– mice has significantly lower suppressive function than that from WT littermates. Left, proliferation assessed by the CFSE dilution measurement of Cd25 − Ths co-cultured with Cd44 high Cd62L low Tregs from Plau −/− or WT mice aged 10 weeks as well as irradiated feeder cells and Cd3 at various ratios of Tregs to Teffs after 3.5 days. Right, comparison of proliferation of Cd25 − Ths co-cultured with Cd44 high Cd62L low Tregs from littermate Plau −/− or WT mice aged 5 weeks as well as irradiated feeder cells and Cd3 at various ratios of Tregs to Teffs after 3.5 days. Max, maximum. Data are representative of 3–5 independent experiments in (E, F, G).
Figure Legend Snippet: Elevated thymic Foxp3 + Treg population while diminished suppressive capability of Cd44 high Cd66L low Tregs in Plau -KO mice. ( A ) Percentages of Cd4 + SP cells among murine thymocytes isolated from wild-type (WT) control mice and Plau heterozygous knockout mice ( Plau +/ − ) assessed by FACS. ( B ) Percentages of Cd25 + Foxp3 + cells among murine Cd4 + SP thymocytes isolated from WT and Plau +/ − mice assessed by FACS. Only gated Cd4 + SP cells from (A) are shown in (B). Data represent one of the four mice per group. ( C ) Comparison of percentages of Cd25 + Foxp3 + cells among murine Cd4 + SP thymocytes between 3-week-old WT and Plau +/ − groups. Data are representative of two independent experiments. ( D ) Left, comparison of percentages of Cd25 + Foxp3 + cells among murine total Cd4 + thymocytes between WT and Plau +/ − and/or Plau −/ − groups, aged 12, 10 and 3 weeks. Error bars represent s.d. values. P -value represented by ‘*' symbols indicate results from a one-tailed Student's t -test for n mice per group (the corresponding number n per group is indicated under each group column). Right, comparison of percentages of Cd25 + Foxp3 + cells among murine total Cd4 + splenocytes between WT, Plau +/ − and Plau −/− groups in 10-week-old mice. ( E ) Comparison of suppressive capability of Cd4 + Cd25 + Tregs between littermate WT, Plau +/ − , and Plau −/− groups, age ≥20 weeks. Left, proliferation assessed by uptake of [H3]thymidine. The P -value above each ratio represented by ‘*' symbols indicates the results from a one-tailed Student's t -test between WT and Plau −/− groups. Right, proliferation assessed by the CFSE dilution measurement of Cd25 − Ths co-cultured with Plau −/− or WT Tregs as well as irradiated feeder cells and Cd3 at various ratios of Tregs to Teffs after 3.5 days. Number in each histogram represents percentage of dividing cells from the total population (the total population indicates the gated DAPI-negative and CD4 + , CFSE stained Ths). ( F ) Left, the gating scheme used to analyze frequencies of Cd44 high Cd62L low Tregs. Right, comparison of percentages of Cd44 high Cd62L low Tregs among total Tregs in spleen (SPL), peripheral LN (pLN) or mesenteric LN (mLN) from WT and Plau +/ − and/or Plau −/ − between groups of mice aged 24 and 3 weeks. The P -value represented by ‘*' symbols indicates the results from a one-tailed Student's t -test. ( G ) Cd44 high Cd62L low Tregs from Plau –/– mice has significantly lower suppressive function than that from WT littermates. Left, proliferation assessed by the CFSE dilution measurement of Cd25 − Ths co-cultured with Cd44 high Cd62L low Tregs from Plau −/− or WT mice aged 10 weeks as well as irradiated feeder cells and Cd3 at various ratios of Tregs to Teffs after 3.5 days. Right, comparison of proliferation of Cd25 − Ths co-cultured with Cd44 high Cd62L low Tregs from littermate Plau −/− or WT mice aged 5 weeks as well as irradiated feeder cells and Cd3 at various ratios of Tregs to Teffs after 3.5 days. Max, maximum. Data are representative of 3–5 independent experiments in (E, F, G).

Techniques Used: Mouse Assay, Isolation, Knock-Out, FACS, One-tailed Test, Cell Culture, Irradiation, Staining

16) Product Images from "Norovirus P Particle Efficiently Elicits Innate, Humoral and Cellular Immunity"

Article Title: Norovirus P Particle Efficiently Elicits Innate, Humoral and Cellular Immunity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063269

Dendritic cell (DC) maturation induced by norovirus P domain complexes. Bone marrow (BM)-derived DCs (BMDCs) were generated as previously described [34] , [35] . BM cells were cultured with 20 ng/ml of GM-CSF and harvested on day 7. This protocol routinely yielded > 90% CD11c + cells as determined by flow cytometry. (A) For maturation assay, BMDCs were stimulated with 10 µg/ml of norovirus VLPs (red), P particles (blue), or P dimers (yellow) for 48 hours, respectively. BMDCs without stimulation (black) were used as control. Cells were harvested at 24 or 48 hours and stained with fluorochrome-conjugated CD11c together with CD40, CD80, CD86 and MHC-II for flowcytometric analysis. (B) ImagestreamX of co-stimulatory and MHC-II molecules were analyzed on BMDC stimulated with AF488-labeled-P particles at 24 hours post stimulation. (C) Kinetic of MHC-II molecule was analyzed on BMDC stimulated with AF488-labeled-P particles at 4, 24 or 48 hours post stimulation. Pictures are representatives of experiments which were repeated three times.
Figure Legend Snippet: Dendritic cell (DC) maturation induced by norovirus P domain complexes. Bone marrow (BM)-derived DCs (BMDCs) were generated as previously described [34] , [35] . BM cells were cultured with 20 ng/ml of GM-CSF and harvested on day 7. This protocol routinely yielded > 90% CD11c + cells as determined by flow cytometry. (A) For maturation assay, BMDCs were stimulated with 10 µg/ml of norovirus VLPs (red), P particles (blue), or P dimers (yellow) for 48 hours, respectively. BMDCs without stimulation (black) were used as control. Cells were harvested at 24 or 48 hours and stained with fluorochrome-conjugated CD11c together with CD40, CD80, CD86 and MHC-II for flowcytometric analysis. (B) ImagestreamX of co-stimulatory and MHC-II molecules were analyzed on BMDC stimulated with AF488-labeled-P particles at 24 hours post stimulation. (C) Kinetic of MHC-II molecule was analyzed on BMDC stimulated with AF488-labeled-P particles at 4, 24 or 48 hours post stimulation. Pictures are representatives of experiments which were repeated three times.

Techniques Used: Derivative Assay, Generated, Cell Culture, Flow Cytometry, Cytometry, Staining, Labeling

17) Product Images from "Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells"

Article Title: Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-25646-8

LMIR8 is highly expressed in pDCs. ( a ) Ba/F3 cells transduced with Flag-tagged LMIR1, LMIR2, LMIR3, LMIR4, LMIR5, LMIR7, LMIR8, or mock were stained with mouse anti-Flag Ab or a control Ab followed by PE-conjugated anti-mouse IgG goat F(ab’) 2 Ab ( upper panel ) or with biotinylated anti-LMIR8 Ab or biotinylated rat IgG2a Ab followed by PE-conjugated streptavidin. ( b–h ) Single cell suspensions were prepared from BM ( b – f ), spleen ( g ), or lymph node ( h ). ( b – d ) Cells were stained with biotinylated anti-LMIR8 Ab or biotin rat IgG2a Ab followed by PE-conjugated streptavidin and FITC-conjugated Abs as indicated. FSC low SSC low populations ( b ), FSC high SSC high populations ( c ), or FSC int SSC int populations ( d ) were gated. ( e – h ), BM cells ( e,f ), spleen cells ( g ), or lymph node cells ( h ) were stained with FITC-conjugated anti-CD11c Ab and APC-conjugated anti-B220 Ab ( e ) or with FITC-conjugated anti-BST2 Ab and APC-conjugated anti-Siglec-H Ab ( f – h ), and then with biotinylated anti-LMIR8 Ab or biotinylated rat IgG2a Ab followed by PE-conjugated streptavidin. CD11c + B220 + ( e ) or BST2 + Siglec-H + ( f – h ) cell populations were gated and analyzed for LMIR8 expression. ( i ) Flt3 ligand-induced BMpDCs, BMmDCs, BMMΦ, or BMMCs were stained with biotinylated anti-LMIR8 Ab or biotinylated rat IgG2a Ab followed by PE-conjugated streptavidin. One representative of four independent experiments is shown.
Figure Legend Snippet: LMIR8 is highly expressed in pDCs. ( a ) Ba/F3 cells transduced with Flag-tagged LMIR1, LMIR2, LMIR3, LMIR4, LMIR5, LMIR7, LMIR8, or mock were stained with mouse anti-Flag Ab or a control Ab followed by PE-conjugated anti-mouse IgG goat F(ab’) 2 Ab ( upper panel ) or with biotinylated anti-LMIR8 Ab or biotinylated rat IgG2a Ab followed by PE-conjugated streptavidin. ( b–h ) Single cell suspensions were prepared from BM ( b – f ), spleen ( g ), or lymph node ( h ). ( b – d ) Cells were stained with biotinylated anti-LMIR8 Ab or biotin rat IgG2a Ab followed by PE-conjugated streptavidin and FITC-conjugated Abs as indicated. FSC low SSC low populations ( b ), FSC high SSC high populations ( c ), or FSC int SSC int populations ( d ) were gated. ( e – h ), BM cells ( e,f ), spleen cells ( g ), or lymph node cells ( h ) were stained with FITC-conjugated anti-CD11c Ab and APC-conjugated anti-B220 Ab ( e ) or with FITC-conjugated anti-BST2 Ab and APC-conjugated anti-Siglec-H Ab ( f – h ), and then with biotinylated anti-LMIR8 Ab or biotinylated rat IgG2a Ab followed by PE-conjugated streptavidin. CD11c + B220 + ( e ) or BST2 + Siglec-H + ( f – h ) cell populations were gated and analyzed for LMIR8 expression. ( i ) Flt3 ligand-induced BMpDCs, BMmDCs, BMMΦ, or BMMCs were stained with biotinylated anti-LMIR8 Ab or biotinylated rat IgG2a Ab followed by PE-conjugated streptavidin. One representative of four independent experiments is shown.

Techniques Used: Transduction, Staining, Expressing

18) Product Images from "Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy"

Article Title: Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2015.45

Generation of B16F10 clones containing TS-MAC or MAC4 . ( a ) PCR analysis with the primers for B16F10 clones containing TS-MAC or MAC4 from microcell-mediated chromosome transfer (MMCT). ( b ) A representative image of FISH analysis of B16F10/TS-MAC#1. Blue indicates 4′,6-diamidino-2-phenylindole signals. The rhodamine (red) signal indicates the centromere sequence of mouse chromosomes. The fluorescein isothiocyanate signal (green), which was observed as a yellow dot, indicates the inserted PAC vector. The yellow arrow shows TS-MAC. An elongated TS-MAC is shown in the inset. ( c ) Flow cytometric analysis of B16F10 clones containing TS-MAC or MAC4 using antibodies against MHC H2-K(d) or -K(b). The gray histogram shows the counts of MHC H2-K(b) polyclonal mouse anti-goat IgG conjugated with allophycocyanin (APC) as a negative control. Red histograms show the counts of MHC H2-K(d) detected by a polyclonal mouse anti-goat IgG conjugated with APC.
Figure Legend Snippet: Generation of B16F10 clones containing TS-MAC or MAC4 . ( a ) PCR analysis with the primers for B16F10 clones containing TS-MAC or MAC4 from microcell-mediated chromosome transfer (MMCT). ( b ) A representative image of FISH analysis of B16F10/TS-MAC#1. Blue indicates 4′,6-diamidino-2-phenylindole signals. The rhodamine (red) signal indicates the centromere sequence of mouse chromosomes. The fluorescein isothiocyanate signal (green), which was observed as a yellow dot, indicates the inserted PAC vector. The yellow arrow shows TS-MAC. An elongated TS-MAC is shown in the inset. ( c ) Flow cytometric analysis of B16F10 clones containing TS-MAC or MAC4 using antibodies against MHC H2-K(d) or -K(b). The gray histogram shows the counts of MHC H2-K(b) polyclonal mouse anti-goat IgG conjugated with allophycocyanin (APC) as a negative control. Red histograms show the counts of MHC H2-K(d) detected by a polyclonal mouse anti-goat IgG conjugated with APC.

Techniques Used: Clone Assay, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Sequencing, Plasmid Preparation, Flow Cytometry, Negative Control

19) Product Images from "TLR4 signals in B lymphocytes are transduced via the B cell antigen receptor and SYK"

Article Title: TLR4 signals in B lymphocytes are transduced via the B cell antigen receptor and SYK

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161117

BCR is required for TLR4-induced activation of SYK. (A) Immunoblot of lysates from wild-type B cells stimulated for various times with LPS (left) or LAS (right). Blots were probed with antibodies to pSYK and SYK. Graphs show mean ± SEM levels of pSYK normalized to SYK or tubulin and then to unstimulated cells (time 0), quantitated from multiple immunoblots. n = 4 (LPS) and 3 (LAS). (B–E) Immunoblots of lysates from IgM + or IgM − B cells (B–D) or Myd88 +/+ or Myd88 −/− B cells (E) stimulated with LPS (B and E), CD40L (C), or LAS (D) for the indicated times. Blots were probed with antibodies to pSYK, pAKT, pERK, SYK, ERK2, and IκBα. Graphs show mean ± SEM levels of pSYK, pAKT, pERK, and IκBα normalized to SYK or ERK2, and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: B, 9 (pSYK except 3 for 15 min), 8 (pAKT), 7 (pERK), and 5 (IκBα); C, 3 (pERK), 3 (IκBα), and 2 (pAKT); D, 3 (pSYK), 4 (pERK), and 4 (IκBα); E, 9 (pSYK) and 7 (pAKT); 2 (pERK and IκBα in WT), 4 (pERK and IκBα in Myd88 −/− ). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using a Mann–Whitney test (A and B, pAKT) or two-way ANOVA (all other graphs; p-values for effect of genotype): *, P
Figure Legend Snippet: BCR is required for TLR4-induced activation of SYK. (A) Immunoblot of lysates from wild-type B cells stimulated for various times with LPS (left) or LAS (right). Blots were probed with antibodies to pSYK and SYK. Graphs show mean ± SEM levels of pSYK normalized to SYK or tubulin and then to unstimulated cells (time 0), quantitated from multiple immunoblots. n = 4 (LPS) and 3 (LAS). (B–E) Immunoblots of lysates from IgM + or IgM − B cells (B–D) or Myd88 +/+ or Myd88 −/− B cells (E) stimulated with LPS (B and E), CD40L (C), or LAS (D) for the indicated times. Blots were probed with antibodies to pSYK, pAKT, pERK, SYK, ERK2, and IκBα. Graphs show mean ± SEM levels of pSYK, pAKT, pERK, and IκBα normalized to SYK or ERK2, and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: B, 9 (pSYK except 3 for 15 min), 8 (pAKT), 7 (pERK), and 5 (IκBα); C, 3 (pERK), 3 (IκBα), and 2 (pAKT); D, 3 (pSYK), 4 (pERK), and 4 (IκBα); E, 9 (pSYK) and 7 (pAKT); 2 (pERK and IκBα in WT), 4 (pERK and IκBα in Myd88 −/− ). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using a Mann–Whitney test (A and B, pAKT) or two-way ANOVA (all other graphs; p-values for effect of genotype): *, P

Techniques Used: Activation Assay, Western Blot, MANN-WHITNEY

SYK transduces TLR4 signals leading to activation of ERK and AKT but not NF-κB. (A–C) Immunoblots of lysates from control and mutant B cells stimulated with LPS (A), CD40L (B), or LAS (C) for the indicated times. Blots were probed with antibodies to phospho-Ser473 AKT (pAKT), phospho-Thr202/Tyr204 ERK1/ERK2 (pERK), SYK, IκBα, and ERK2. Graphs show mean ± SEM levels of pAKT, pERK, and IκBα normalized to ERK2 and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: A, 3 (pAKT), 9 (pERK), and 3 (IκBα); B, 3 (pAKT), 7 (pERK), and 6 (IκBα); C, 4 (pERK) and 4 (IκBα). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype): *, P
Figure Legend Snippet: SYK transduces TLR4 signals leading to activation of ERK and AKT but not NF-κB. (A–C) Immunoblots of lysates from control and mutant B cells stimulated with LPS (A), CD40L (B), or LAS (C) for the indicated times. Blots were probed with antibodies to phospho-Ser473 AKT (pAKT), phospho-Thr202/Tyr204 ERK1/ERK2 (pERK), SYK, IκBα, and ERK2. Graphs show mean ± SEM levels of pAKT, pERK, and IκBα normalized to ERK2 and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: A, 3 (pAKT), 9 (pERK), and 3 (IκBα); B, 3 (pAKT), 7 (pERK), and 6 (IκBα); C, 4 (pERK) and 4 (IκBα). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype): *, P

Techniques Used: Activation Assay, Western Blot, Mutagenesis

20) Product Images from "Chronic Systemic Exposure to Low-Dose Rotenone Induced Central and Peripheral Neuropathology and Motor Deficits in Mice: Reproducible Animal Model of Parkinson’s Disease"

Article Title: Chronic Systemic Exposure to Low-Dose Rotenone Induced Central and Peripheral Neuropathology and Motor Deficits in Mice: Reproducible Animal Model of Parkinson’s Disease

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21093254

Chronic exposure to low-dose rotenone induced α-synuclein (α-Syn) accumulation in dopaminergic neurons in the SNpc of mice. Representative photomicrographs of TH and α-Syn double immunostaining in the SNpc of mice. Red: TH-positive neurons. Green: α-Syn. Blue: nuclear staining with Hoechst 33342. Solid arrowheads: increased α-Syn signals in TH-positive neuronal soma. Open arrowheads: α-Syn-positive neurite. Scale bar = 20 µm.
Figure Legend Snippet: Chronic exposure to low-dose rotenone induced α-synuclein (α-Syn) accumulation in dopaminergic neurons in the SNpc of mice. Representative photomicrographs of TH and α-Syn double immunostaining in the SNpc of mice. Red: TH-positive neurons. Green: α-Syn. Blue: nuclear staining with Hoechst 33342. Solid arrowheads: increased α-Syn signals in TH-positive neuronal soma. Open arrowheads: α-Syn-positive neurite. Scale bar = 20 µm.

Techniques Used: Mouse Assay, Double Immunostaining, Staining

Changes in α-Syn-positive signals in the hypoglossal nucleus of rotenone-treated mice. Representative photomicrographs of ChAT and α-Syn double immunostaining. Red: ChAT-positive neurons. Green: α-Syn. Blue: nuclear staining with Hoechst 33342. Scale bar = 20 µm.
Figure Legend Snippet: Changes in α-Syn-positive signals in the hypoglossal nucleus of rotenone-treated mice. Representative photomicrographs of ChAT and α-Syn double immunostaining. Red: ChAT-positive neurons. Green: α-Syn. Blue: nuclear staining with Hoechst 33342. Scale bar = 20 µm.

Techniques Used: Mouse Assay, Double Immunostaining, Staining

Chronic exposure to low-dose rotenone induced α-Syn accumulation in the intestinal myenteric plexus of mice. Representative photomicrographs of Tubulin β III and α-Syn double immunostaining. Green: Tubulin β III. Red: α-Syn. Blue: nuclear staining with Hoechst 33342. (Left, Center) Scale bar = 20 µm. (Right) Magnified photographs of white boxes. Scale bar = 10 µm.
Figure Legend Snippet: Chronic exposure to low-dose rotenone induced α-Syn accumulation in the intestinal myenteric plexus of mice. Representative photomicrographs of Tubulin β III and α-Syn double immunostaining. Green: Tubulin β III. Red: α-Syn. Blue: nuclear staining with Hoechst 33342. (Left, Center) Scale bar = 20 µm. (Right) Magnified photographs of white boxes. Scale bar = 10 µm.

Techniques Used: Mouse Assay, Double Immunostaining, Staining

Chronic exposure to low-dose rotenone induced α-Syn accumulation in the cholinergic neurons in the DMV of mice. Representative photomicrographs of ChAT and α-Syn double immunostaining. Red: ChAT-positive neurons. Green: α-Syn. Blue: nuclear staining with Hoechst 33342. Solid arrowheads: increased α-Syn signals in neuronal soma in the DMV. Arrows: α-Syn-negative neurons in the hypoglossal nucleus. Scale bar = 20 µm.
Figure Legend Snippet: Chronic exposure to low-dose rotenone induced α-Syn accumulation in the cholinergic neurons in the DMV of mice. Representative photomicrographs of ChAT and α-Syn double immunostaining. Red: ChAT-positive neurons. Green: α-Syn. Blue: nuclear staining with Hoechst 33342. Solid arrowheads: increased α-Syn signals in neuronal soma in the DMV. Arrows: α-Syn-negative neurons in the hypoglossal nucleus. Scale bar = 20 µm.

Techniques Used: Mouse Assay, Double Immunostaining, Staining

21) Product Images from "Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer"

Article Title: Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000203

5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P
Figure Legend Snippet: 5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P

Techniques Used: Construct, Transduction, Derivative Assay

22) Product Images from "Alanine Scanning Mutagenesis of the C-Terminal Cytosolic End of Gpm6a Identifies Key Residues Essential for the Formation of Filopodia"

Article Title: Alanine Scanning Mutagenesis of the C-Terminal Cytosolic End of Gpm6a Identifies Key Residues Essential for the Formation of Filopodia

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00314

Deletion of the N- or the C-terminal intracellular domain diminishes the amount of Gpm6a on cell surface. (A) Flow cytometry was used to measure and calculate the mean fluorescence intensity of EGFP in the population of EGFP-positive N2a cells transfected with the indicated vectors as a measure of the total amount of EGFP-tagged proteins. Data are means ± SEM of two independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects revealed no significant differences between Gpm6a wt-EGFP vs Gpm6a ΔN-EGFP and Gpm6a wt-EGFP vs Gpm6a ΔC-EGFP. (B) The mean fluorescence intensity of the surface-labeled Gpm6a measured by flow cytometry in the population of EGFP-positive N2a cells transfected with the indicated vectors. Surface Gpm6a was labeleld by immunostaining of non-permeabilized cells with the rat anti-Gpm6a antibody followed by goat anti-rat IgG conjugated to Alexa Fluor 647. Data are means ± SEM of two independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects, ∗∗∗ p
Figure Legend Snippet: Deletion of the N- or the C-terminal intracellular domain diminishes the amount of Gpm6a on cell surface. (A) Flow cytometry was used to measure and calculate the mean fluorescence intensity of EGFP in the population of EGFP-positive N2a cells transfected with the indicated vectors as a measure of the total amount of EGFP-tagged proteins. Data are means ± SEM of two independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects revealed no significant differences between Gpm6a wt-EGFP vs Gpm6a ΔN-EGFP and Gpm6a wt-EGFP vs Gpm6a ΔC-EGFP. (B) The mean fluorescence intensity of the surface-labeled Gpm6a measured by flow cytometry in the population of EGFP-positive N2a cells transfected with the indicated vectors. Surface Gpm6a was labeleld by immunostaining of non-permeabilized cells with the rat anti-Gpm6a antibody followed by goat anti-rat IgG conjugated to Alexa Fluor 647. Data are means ± SEM of two independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects, ∗∗∗ p

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Transfection, Labeling, Immunostaining

Substitution with alanine of K250, D253/K255, and E258/E259 diminishes the amount of Gpm6a on cell surface. (A) Flow cytometry was used to measure and calculate the mean fluorescence intensity of EGFP in the population of EGFP-positive N2a cells transfected with the indicated vectors as a measure of the total amount of EGFP-tagged proteins. Data are means ± SEM of three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects revealed no significant differences between Gpm6a wt-EGFP vs K250A-EGFP, Gpm6a wt-EGFP vs D253A/K255A-EGFP, and Gpm6a wt-EGFP vs E258A/E259A-EGFP. (B) The mean fluorescence intensity of the surface-labeled Gpm6a measured by flow cytometry in the population of EGFP-positive N2a cells transfected with the indicated vectors. Surface Gpm6a was labeleld by immunostaining of non-permeabilized cells with the rat anti-Gpm6a antibody followed by goat anti-rat IgG conjugated to Alexa Fluor 647. Data are means ± SEM of three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects, ∗∗∗ p
Figure Legend Snippet: Substitution with alanine of K250, D253/K255, and E258/E259 diminishes the amount of Gpm6a on cell surface. (A) Flow cytometry was used to measure and calculate the mean fluorescence intensity of EGFP in the population of EGFP-positive N2a cells transfected with the indicated vectors as a measure of the total amount of EGFP-tagged proteins. Data are means ± SEM of three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects revealed no significant differences between Gpm6a wt-EGFP vs K250A-EGFP, Gpm6a wt-EGFP vs D253A/K255A-EGFP, and Gpm6a wt-EGFP vs E258A/E259A-EGFP. (B) The mean fluorescence intensity of the surface-labeled Gpm6a measured by flow cytometry in the population of EGFP-positive N2a cells transfected with the indicated vectors. Surface Gpm6a was labeleld by immunostaining of non-permeabilized cells with the rat anti-Gpm6a antibody followed by goat anti-rat IgG conjugated to Alexa Fluor 647. Data are means ± SEM of three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test for post hoc effects, ∗∗∗ p

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Transfection, Labeling, Immunostaining

23) Product Images from "Tuning T Cell Signaling Sensitivity Alters the Behavior of CD4+ T Cells During an Immune Response"

Article Title: Tuning T Cell Signaling Sensitivity Alters the Behavior of CD4+ T Cells During an Immune Response

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1701422

TCR sensitization via Scn5a expression correlates with more rapid and robust TCR-proximal signaling, using PLCγ phosphorylation and Ca2+ flux as readouts (A) Phosphorylation of PLCγ (relative to b-actin) in response to sub-maximal CD3 crosslinking (10µg/mL) was measured at 30 sec., 1 min., 3 min., 5 min., 10 min. and 15 min., in 1.5×10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. Fold change (a ratio of Scn5a+CD4-Cre+ to Scn5a+CD4-Cre- pPLCγ:b-actin maximum levels) is quantified on the right. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts. (B) Ca2+ flux was measured in 10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. CD3e/IgG complexes were added at 5 minutes, except in the control wells, in which Ca2+ buffer alone was used. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts.
Figure Legend Snippet: TCR sensitization via Scn5a expression correlates with more rapid and robust TCR-proximal signaling, using PLCγ phosphorylation and Ca2+ flux as readouts (A) Phosphorylation of PLCγ (relative to b-actin) in response to sub-maximal CD3 crosslinking (10µg/mL) was measured at 30 sec., 1 min., 3 min., 5 min., 10 min. and 15 min., in 1.5×10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. Fold change (a ratio of Scn5a+CD4-Cre+ to Scn5a+CD4-Cre- pPLCγ:b-actin maximum levels) is quantified on the right. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts. (B) Ca2+ flux was measured in 10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. CD3e/IgG complexes were added at 5 minutes, except in the control wells, in which Ca2+ buffer alone was used. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts.

Techniques Used: Expressing, Size-exclusion Chromatography, Mouse Assay

24) Product Images from "Loss of signaling via Gα13 in germinal center B cell-derived lymphoma"

Article Title: Loss of signaling via Gα13 in germinal center B cell-derived lymphoma

Journal: Nature

doi: 10.1038/nature13765

P2RY8-dependent suppression of GC B cell survival and promotion of B cell confinement to the GC niche is receptor specific (a) Transwell migration of WEHI231 cells transduced with retrovirus encoding the control Gα13-coupled receptor, Tbxa2r, toward CXCL12 (100 ng/mL) in the presence or absence of the thromboxane A2 analogue, U-46619. (b, c) Fold change in frequency of Thy1.1 reporter + GC relative to Fo B cells of PPs (b) or mLN (c) from BM chimeras reconstituted with S1pr2 KO BM transduced with empty vector (control) or Tbxa2r. (d) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Gpr183 +/- B cells transduced with empty vector, Tbxa2r or P2RY8, and assessed 24 h after cell transfer. Scale bar is 200 μm. Data in a and d are one experiment representative of 2. Data in b and c are from one experiment (n=4 in each group). ** P
Figure Legend Snippet: P2RY8-dependent suppression of GC B cell survival and promotion of B cell confinement to the GC niche is receptor specific (a) Transwell migration of WEHI231 cells transduced with retrovirus encoding the control Gα13-coupled receptor, Tbxa2r, toward CXCL12 (100 ng/mL) in the presence or absence of the thromboxane A2 analogue, U-46619. (b, c) Fold change in frequency of Thy1.1 reporter + GC relative to Fo B cells of PPs (b) or mLN (c) from BM chimeras reconstituted with S1pr2 KO BM transduced with empty vector (control) or Tbxa2r. (d) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Gpr183 +/- B cells transduced with empty vector, Tbxa2r or P2RY8, and assessed 24 h after cell transfer. Scale bar is 200 μm. Data in a and d are one experiment representative of 2. Data in b and c are from one experiment (n=4 in each group). ** P

Techniques Used: Migration, Transduction, Plasmid Preparation, Immunohistochemistry, Mouse Assay

Lymphoma-associated S1PR2 mutations are functionally disruptive and loss of Gα13 is sufficient to promote GC B cell survival and lymphomagenesis (a) Schematic of S1PR2 with mutated residues highlighted. Circles denote mutated residues conserved in S1PR2 across species, filled circles, conserved across Type A GPCRs, squares, residues not conserved across species, and asterisk, position of truncating frameshift mutation. (b) Western blot of FLAG expression in WEHI231 cells transduced with FLAG-tagged WT or mutant S1PR2 or empty vector. Shown is one experiment representative of 3 independent biologic replicates. The gap in the gel image marks the position of one lane that was not relevant to this experiment and was removed for clarity. (c) WEHI231 cells transduced as in b were stimulated with CXCL12 (100 ng/ml) in the presence or absence of S1P (1 nM) for 5 minutes and analyzed for phosphorylation of Akt (pAkt S473) by intracellular FACS. Shown is MFI of pAkt in samples treated with both CXCL12 and S1P relative to CXCL12 alone. Data are pooled from 4 independent experiments. (d) Transwell migration of cells transduced as in b, in response to CXCL12 (100 ng/ml) in the presence or absence of S1P (1 nM). Shown is the relative migration of transduced cells to CXCL12 in the presence versus absence of S1P. Data are pooled from 8 independent experiments. (e) Percentages of CD45.2 follicular B cells (Fo) and GC B cells from mLNs of mixed BM chimeras generated with ∼70% WT CD45.1 cells and ∼30% S1pr2 WT (n=9), heterozygous (n=28) or knockout (n=19) CD45.2 BM, assessed by FACS. Gating scheme is shown in Extended Data Fig 3a . Data are pooled from 4 independent experiments. (f) Fold change in frequency of Thy1.1 reporter + cells in GC relative to Fo B cells of PPs from chimeras reconstituted with S1pr2 +/- BM transduced with retrovirus expressing either WT (n=17) or mutant S1PR2 (R147C, n=8; R329C, n=6). Gating scheme is shown in Extended Data Fig. 3c . Data are pooled from 3 independent experiments. (g) Percentages of CD45.2 + Fo and GC B cells from mLNs of mixed BM chimeras generated with ∼40% Gna13 WT ( f /+) (n=12) or KO ( f/f mb1-cre ) (n=17) CD45.2 cells and ∼60% WT CD45.1 cells. Data are pooled from 4 independent experiments. (h, i) Intracellular FACS for pAkt in GC B cells from mLN of Gna13 (h) or Arhgef1 (i) mixed BM chimeras that were stimulated ex vivo with or without CXCL12 (300 ng/ml) in the presence or absence of S1P (10 nM) for 10 minutes. Data in graphs are mean +/- SEM and are from one experiment with 3 biologic replicates for each treatment and are representative of 4 experiments ( Gna13 ) or 3 experiments ( Arhgef1 ) (j) FACS analysis of mLN of 1 year-old Gna13 WT or Gna13 KO (#307). Percent of total cells that are GC B cells is indicated. (k) GC B cell number from mLN of Gna13 WT and heterozygous (n=20) or KO (n=18) animals aged to 12 to 16 months. (l) Gross appearance of mLN and spleen from Gna13 WT control and 2 Gna13 KO animals. Arrow in #307 denotes splenic nodule (see also Extended Data Fig. 4c-e ). Scale bar is 1 cm. * P
Figure Legend Snippet: Lymphoma-associated S1PR2 mutations are functionally disruptive and loss of Gα13 is sufficient to promote GC B cell survival and lymphomagenesis (a) Schematic of S1PR2 with mutated residues highlighted. Circles denote mutated residues conserved in S1PR2 across species, filled circles, conserved across Type A GPCRs, squares, residues not conserved across species, and asterisk, position of truncating frameshift mutation. (b) Western blot of FLAG expression in WEHI231 cells transduced with FLAG-tagged WT or mutant S1PR2 or empty vector. Shown is one experiment representative of 3 independent biologic replicates. The gap in the gel image marks the position of one lane that was not relevant to this experiment and was removed for clarity. (c) WEHI231 cells transduced as in b were stimulated with CXCL12 (100 ng/ml) in the presence or absence of S1P (1 nM) for 5 minutes and analyzed for phosphorylation of Akt (pAkt S473) by intracellular FACS. Shown is MFI of pAkt in samples treated with both CXCL12 and S1P relative to CXCL12 alone. Data are pooled from 4 independent experiments. (d) Transwell migration of cells transduced as in b, in response to CXCL12 (100 ng/ml) in the presence or absence of S1P (1 nM). Shown is the relative migration of transduced cells to CXCL12 in the presence versus absence of S1P. Data are pooled from 8 independent experiments. (e) Percentages of CD45.2 follicular B cells (Fo) and GC B cells from mLNs of mixed BM chimeras generated with ∼70% WT CD45.1 cells and ∼30% S1pr2 WT (n=9), heterozygous (n=28) or knockout (n=19) CD45.2 BM, assessed by FACS. Gating scheme is shown in Extended Data Fig 3a . Data are pooled from 4 independent experiments. (f) Fold change in frequency of Thy1.1 reporter + cells in GC relative to Fo B cells of PPs from chimeras reconstituted with S1pr2 +/- BM transduced with retrovirus expressing either WT (n=17) or mutant S1PR2 (R147C, n=8; R329C, n=6). Gating scheme is shown in Extended Data Fig. 3c . Data are pooled from 3 independent experiments. (g) Percentages of CD45.2 + Fo and GC B cells from mLNs of mixed BM chimeras generated with ∼40% Gna13 WT ( f /+) (n=12) or KO ( f/f mb1-cre ) (n=17) CD45.2 cells and ∼60% WT CD45.1 cells. Data are pooled from 4 independent experiments. (h, i) Intracellular FACS for pAkt in GC B cells from mLN of Gna13 (h) or Arhgef1 (i) mixed BM chimeras that were stimulated ex vivo with or without CXCL12 (300 ng/ml) in the presence or absence of S1P (10 nM) for 10 minutes. Data in graphs are mean +/- SEM and are from one experiment with 3 biologic replicates for each treatment and are representative of 4 experiments ( Gna13 ) or 3 experiments ( Arhgef1 ) (j) FACS analysis of mLN of 1 year-old Gna13 WT or Gna13 KO (#307). Percent of total cells that are GC B cells is indicated. (k) GC B cell number from mLN of Gna13 WT and heterozygous (n=20) or KO (n=18) animals aged to 12 to 16 months. (l) Gross appearance of mLN and spleen from Gna13 WT control and 2 Gna13 KO animals. Arrow in #307 denotes splenic nodule (see also Extended Data Fig. 4c-e ). Scale bar is 1 cm. * P

Techniques Used: Mutagenesis, Western Blot, Expressing, Transduction, Plasmid Preparation, FACS, Migration, Generated, Knock-Out, Ex Vivo

S1PR2 heterozygosity confers a survival advantage to GC B cells and R147C S1PR2 fails to function (a, b) Flow cytometry of Fo and GC B cells from mLN and PP of mixed BM chimeras generated as in Fig. 1e . Gating strategy for FoB and GCB in mLN is shown in a and percentages of CD45.2 + cells in Fo and GC B cells from PP are shown in b. Data in b are pooled from 4 independent experiments. (c) Gating strategy of Thy1.1 reporter expression in Fo and GC B cells from PP (c) or fold change in Thy1.1 + cells in GC relative to Fo B cells of mLN (d) of retrovirally transduced BM chimeras as described in Fig. 1f . Data in d are pooled from 3 independent experiments. * P
Figure Legend Snippet: S1PR2 heterozygosity confers a survival advantage to GC B cells and R147C S1PR2 fails to function (a, b) Flow cytometry of Fo and GC B cells from mLN and PP of mixed BM chimeras generated as in Fig. 1e . Gating strategy for FoB and GCB in mLN is shown in a and percentages of CD45.2 + cells in Fo and GC B cells from PP are shown in b. Data in b are pooled from 4 independent experiments. (c) Gating strategy of Thy1.1 reporter expression in Fo and GC B cells from PP (c) or fold change in Thy1.1 + cells in GC relative to Fo B cells of mLN (d) of retrovirally transduced BM chimeras as described in Fig. 1f . Data in d are pooled from 3 independent experiments. * P

Techniques Used: Flow Cytometry, Cytometry, Generated, Expressing

P2RY8, mutated in GCB-DLBCL and BL, suppresses GC B cell growth and promotes B cell confinement via Gα 13 (a) Schematic of P2RY8 with locations of mutated residues in GCB-DLBCL and BL. Residues are marked as for S1PR2 in Figure 1a . (b) Phylogenetic tree of P2RY8 across species. (c) Quantitative PCR of S1PR1 , S1PR2 and P2RY8 in FACS-sorted human tonsillar Fo and GC B cells. Data in c are from 5 donors. (d, e) Fold change in frequency of Thy1.1 reporter + cells in GC relative to Fo B cells of PPs from BM chimeras reconstituted with S1pr2 KO BM (d) or Gna13 KO ( f/f mb1-cre ) BM (e) transduced with retrovirus expressing P2RY8, or with S1PR2, GNA13 or R147C mutant S1PR2 (control). Data in d are pooled from 2 independent experiments (S1PR2, n=4; Control, n=8; P2RY8, n=8). Data in e are from one experiment (n=4 in each group). (f, g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (f) or Gna13 WT or KO (g) B cells transduced with retroviral vector encoding Thy1.1 alone (vector) or P2RY8 and Thy1.1, assessed 24 h after cell transfer. Scale bar is 200 μm in f and g. Data in f are representative of 3 and in g of 2 independent experiments. * P
Figure Legend Snippet: P2RY8, mutated in GCB-DLBCL and BL, suppresses GC B cell growth and promotes B cell confinement via Gα 13 (a) Schematic of P2RY8 with locations of mutated residues in GCB-DLBCL and BL. Residues are marked as for S1PR2 in Figure 1a . (b) Phylogenetic tree of P2RY8 across species. (c) Quantitative PCR of S1PR1 , S1PR2 and P2RY8 in FACS-sorted human tonsillar Fo and GC B cells. Data in c are from 5 donors. (d, e) Fold change in frequency of Thy1.1 reporter + cells in GC relative to Fo B cells of PPs from BM chimeras reconstituted with S1pr2 KO BM (d) or Gna13 KO ( f/f mb1-cre ) BM (e) transduced with retrovirus expressing P2RY8, or with S1PR2, GNA13 or R147C mutant S1PR2 (control). Data in d are pooled from 2 independent experiments (S1PR2, n=4; Control, n=8; P2RY8, n=8). Data in e are from one experiment (n=4 in each group). (f, g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (f) or Gna13 WT or KO (g) B cells transduced with retroviral vector encoding Thy1.1 alone (vector) or P2RY8 and Thy1.1, assessed 24 h after cell transfer. Scale bar is 200 μm in f and g. Data in f are representative of 3 and in g of 2 independent experiments. * P

Techniques Used: Real-time Polymerase Chain Reaction, FACS, Transduction, Expressing, Mutagenesis, Immunohistochemistry, Mouse Assay, Transgenic Assay, Plasmid Preparation

Lymphoma-associated mutations result in loss of expression and function of S1PR2 (a-c) Surface expression of FLAG (a), quantitative PCR of human S1PR2 (b) or Thy1.1 reporter expression (c) in mouse WEHI231 B lymphoma cells transduced as described in Fig. 1b . Shown in a are histograms of transduced cells (Thy1.1 + ) in blue and untransduced cells (Thy1.1 − ) in gray. 5 of 8 S1PR2 mutations showed loss of protein expression despite strong transcript and reporter expression. Loss of expression in these 5 mutants was likely a result of degradation of improperly folded proteins in the ER. (d) Representative FACS plots of transwell migration of WEHI231 cells transduced with vector, WT or R147C mutant S1PR2 to the indicated stimuli or the input sample. Numbers indicate % of cells positive for the Thy1.1 reporter. (e) WEHI231 cells stimulated as in Fig. 1d were analyzed for phosphorylation of Akt (pAkt S473) by Western blot or by intracellular FACS. Data in a and c are representative of 4 independent experiments and d and e of 3 independent experiments.
Figure Legend Snippet: Lymphoma-associated mutations result in loss of expression and function of S1PR2 (a-c) Surface expression of FLAG (a), quantitative PCR of human S1PR2 (b) or Thy1.1 reporter expression (c) in mouse WEHI231 B lymphoma cells transduced as described in Fig. 1b . Shown in a are histograms of transduced cells (Thy1.1 + ) in blue and untransduced cells (Thy1.1 − ) in gray. 5 of 8 S1PR2 mutations showed loss of protein expression despite strong transcript and reporter expression. Loss of expression in these 5 mutants was likely a result of degradation of improperly folded proteins in the ER. (d) Representative FACS plots of transwell migration of WEHI231 cells transduced with vector, WT or R147C mutant S1PR2 to the indicated stimuli or the input sample. Numbers indicate % of cells positive for the Thy1.1 reporter. (e) WEHI231 cells stimulated as in Fig. 1d were analyzed for phosphorylation of Akt (pAkt S473) by Western blot or by intracellular FACS. Data in a and c are representative of 4 independent experiments and d and e of 3 independent experiments.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, FACS, Migration, Transduction, Plasmid Preparation, Mutagenesis, Western Blot

Human P2RY8 suppresses GC B cell growth and promotes B cell confinement to the GC in mice (a, b) P2RY8 mutations arising in GCB-DLBCL and BL disrupt receptor expression. Flag-tagged versions of six point mutant and the wild-type receptor were expressed in WEH231 B cells and surface expression examined by FLAG flow cytometry (a). The transduction efficiency of each construct was confirmed to be similar based on IRES-Thy 1.1 reporter expression (b). (c, d) Fold change in Thy1.1 reporter + GC relative to Fo B cells from mLN of chimeras described in Fig. 4d-e . (e-g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (e), Gpr183 +/- (f) or Gna13 WT or KO (g) B cells transduced as in Fig. 4f and g and assessed 24 h after cell transfer. Data in e and g are additional examples of the experiments shown in Fig. 4f and g , respectively. Data in f are representative of 4 independent experiments. Scale bar is 200 μm in e-g. * P
Figure Legend Snippet: Human P2RY8 suppresses GC B cell growth and promotes B cell confinement to the GC in mice (a, b) P2RY8 mutations arising in GCB-DLBCL and BL disrupt receptor expression. Flag-tagged versions of six point mutant and the wild-type receptor were expressed in WEH231 B cells and surface expression examined by FLAG flow cytometry (a). The transduction efficiency of each construct was confirmed to be similar based on IRES-Thy 1.1 reporter expression (b). (c, d) Fold change in Thy1.1 reporter + GC relative to Fo B cells from mLN of chimeras described in Fig. 4d-e . (e-g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (e), Gpr183 +/- (f) or Gna13 WT or KO (g) B cells transduced as in Fig. 4f and g and assessed 24 h after cell transfer. Data in e and g are additional examples of the experiments shown in Fig. 4f and g , respectively. Data in f are representative of 4 independent experiments. Scale bar is 200 μm in e-g. * P

Techniques Used: Mouse Assay, Expressing, Mutagenesis, Flow Cytometry, Cytometry, Transduction, Construct, Immunohistochemistry, Transgenic Assay

25) Product Images from "A DRUG-BASED CELLULAR ASSAY (DBCA) FOR STUDYING CYTOTOXIC AND CYTOPROTECTIVE ACTIVITIES OF THE PRION PROTEIN: A PRACTICAL GUIDE"

Article Title: A DRUG-BASED CELLULAR ASSAY (DBCA) FOR STUDYING CYTOTOXIC AND CYTOPROTECTIVE ACTIVITIES OF THE PRION PROTEIN: A PRACTICAL GUIDE

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2010.11.005

Slot blot-based assay for γ-H2AX
Figure Legend Snippet: Slot blot-based assay for γ-H2AX

Techniques Used: Dot Blot

γ -H2AX assay. Cells expressing WT or ΔCR PrP were treated with 500 μg/ml Zeocin for 2 hours, and DNA damage was detected by slot blot using an antibody against γ-H2AX. The γ-H2AX signals (right-hand panels) were
Figure Legend Snippet: γ -H2AX assay. Cells expressing WT or ΔCR PrP were treated with 500 μg/ml Zeocin for 2 hours, and DNA damage was detected by slot blot using an antibody against γ-H2AX. The γ-H2AX signals (right-hand panels) were

Techniques Used: Expressing, Dot Blot

26) Product Images from "Genome protective effect of metformin as revealed by reduced level of constitutive DNA damage signaling"

Article Title: Genome protective effect of metformin as revealed by reduced level of constitutive DNA damage signaling

Journal: Aging (Albany NY)

doi:

Effect of metformin (MF) on the level of constitutive γH2AX expression in A549 cells Exponentially growing A549 cells were left untreated (Ctrl) or treated with 1, 5 or 20 mM metformin for 48 h. Left panels present bivariate distributions of cellular DNA content versus intensity of γH2AX immunofluorescence (IF) detected with H2AX-Ser139 phospho-specific Ab in cells of these cultures; fluorescence of individual cells was measured by laser scanning cytometry (LSC) [ 76 ]. Based on differences in DNA content the cells were gated in G 1 , S and G 2 M phases of the cell cycle, as shown in the left panel, and the mean values of γH2AX IF for cells in each of these cell cycle phases by were obtained gating analysis. These mean values (+SD) are presented as the bar plots (right panel). The percent decrease in mean values of γH2AX expression of the metformin-treated cells with respect to the same phase of the cell cycle of the untreated cells is shown above the respective bars. The skewed dash line shows the upper level of γH2AX IF intensity for 97% of G 1 - and S- phase cells in Ctrl. The insets show cellular DNA content frequency histograms in the respective cultures.
Figure Legend Snippet: Effect of metformin (MF) on the level of constitutive γH2AX expression in A549 cells Exponentially growing A549 cells were left untreated (Ctrl) or treated with 1, 5 or 20 mM metformin for 48 h. Left panels present bivariate distributions of cellular DNA content versus intensity of γH2AX immunofluorescence (IF) detected with H2AX-Ser139 phospho-specific Ab in cells of these cultures; fluorescence of individual cells was measured by laser scanning cytometry (LSC) [ 76 ]. Based on differences in DNA content the cells were gated in G 1 , S and G 2 M phases of the cell cycle, as shown in the left panel, and the mean values of γH2AX IF for cells in each of these cell cycle phases by were obtained gating analysis. These mean values (+SD) are presented as the bar plots (right panel). The percent decrease in mean values of γH2AX expression of the metformin-treated cells with respect to the same phase of the cell cycle of the untreated cells is shown above the respective bars. The skewed dash line shows the upper level of γH2AX IF intensity for 97% of G 1 - and S- phase cells in Ctrl. The insets show cellular DNA content frequency histograms in the respective cultures.

Techniques Used: Expressing, Immunofluorescence, Fluorescence, Cytometry

27) Product Images from "Relevance of Caspase-1 and Nlrp3 Inflammasome on Inflammatory Bone Resorption in A Murine Model of Periodontitis"

Article Title: Relevance of Caspase-1 and Nlrp3 Inflammasome on Inflammatory Bone Resorption in A Murine Model of Periodontitis

Journal: Scientific Reports

doi: 10.1038/s41598-020-64685-y

( A ) Representative images of H/E-stained sections of each experimental group (non-disease control/PBS-injected or diseased/Aa-injected) according to the genotype (WT, Nlrp3-KO or Casp1-KO) at 100X magnification (BC, bone crest, R, palatal root of the first molar, * indicates inflammation in the injection area) ( B ) Representative images of immunofluorescence detection of the pan-leukocyte marker CD45 and the neutrophil marker Ly6G in the gingival tissues of WT, Nlrp3 and Casp1-KO mice, according to the experimental condition (non-disease control/PBS-injected or diseased/Aa-injected). Nuclei were counterstained with DAPI. The results for the quantitation of mean fluorescence intensity (MFI) in the red channel (AlexaFluor 594) of CD45 or Ly6G according to the experimental condition and genotype are presented in the graphs. Nine semi-serial sections from each animal and experimental condition spanning 900 µm in the sagittal (antero-posterior) plane. Bars represent means and vertical lines the standard deviation of MFI values from at least 4 animals per group and experimental condition (Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s multiple comparisons test).
Figure Legend Snippet: ( A ) Representative images of H/E-stained sections of each experimental group (non-disease control/PBS-injected or diseased/Aa-injected) according to the genotype (WT, Nlrp3-KO or Casp1-KO) at 100X magnification (BC, bone crest, R, palatal root of the first molar, * indicates inflammation in the injection area) ( B ) Representative images of immunofluorescence detection of the pan-leukocyte marker CD45 and the neutrophil marker Ly6G in the gingival tissues of WT, Nlrp3 and Casp1-KO mice, according to the experimental condition (non-disease control/PBS-injected or diseased/Aa-injected). Nuclei were counterstained with DAPI. The results for the quantitation of mean fluorescence intensity (MFI) in the red channel (AlexaFluor 594) of CD45 or Ly6G according to the experimental condition and genotype are presented in the graphs. Nine semi-serial sections from each animal and experimental condition spanning 900 µm in the sagittal (antero-posterior) plane. Bars represent means and vertical lines the standard deviation of MFI values from at least 4 animals per group and experimental condition (Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s multiple comparisons test).

Techniques Used: Staining, Injection, Immunofluorescence, Marker, Mouse Assay, Quantitation Assay, Fluorescence, Standard Deviation

28) Product Images from "The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells"

Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02765

Morphological alterations induced by A. hydrophila ATCC ® 7966 TM OMVs in PBMCs. (A) After 2 h post-stimulation with A. hydrophila OMVs, no morphology changes of the PBMCs were observed compared with PBMCs after 24 h post-stimulation with vesicles, after 24 h post-stimulation conglomerated cells were observed (arrowheads) (B) . Giemsa staining revealed differences in the affinity of the dye to the nucleus and cytoplasm, bigger cells, and vacuolization of the stimulated PBMCs with A. hydrophila OMVs (arrowheads) (C) . Confocal microscopy, showed defective tubulin polymerization in PBMCs stimulated with OMVs (D) . bars = 50 μm.
Figure Legend Snippet: Morphological alterations induced by A. hydrophila ATCC ® 7966 TM OMVs in PBMCs. (A) After 2 h post-stimulation with A. hydrophila OMVs, no morphology changes of the PBMCs were observed compared with PBMCs after 24 h post-stimulation with vesicles, after 24 h post-stimulation conglomerated cells were observed (arrowheads) (B) . Giemsa staining revealed differences in the affinity of the dye to the nucleus and cytoplasm, bigger cells, and vacuolization of the stimulated PBMCs with A. hydrophila OMVs (arrowheads) (C) . Confocal microscopy, showed defective tubulin polymerization in PBMCs stimulated with OMVs (D) . bars = 50 μm.

Techniques Used: Staining, Confocal Microscopy

29) Product Images from "Role of PAR-4 in ovarian cancer"

Article Title: Role of PAR-4 in ovarian cancer

Journal: Oncotarget

doi:

Effect of PAR-4 levels on cell apoptosis A. Analysis of caspase 3/7 activity. SKOV-3 cells are transfected with control or PAR-4 expressing plasmid (a) and with control or PAR-4 siRNA (b) and treated or not with 100 nM of taxol. After 24 hours, luminescent assay is performed to measure caspase3/7 activity. Quantitative results are presented in Figures a and b. B. Apoptosis scoring of active caspase-3. SKOV-3 cells are transfected with control or PAR-4 expressing plasmid. 24 hours after, SKOV-3 cells are seeded and treated or not with 100 nM of taxol for 24 hours. SKOV-3 cells are washed, fixed and then, incubated with anti-PAR-4 antibodies (R-334) and active caspase-3 antibodies. Then, cells are incubated for 1 hour with Alexa Fluor-488 donkey anti-rabbit IgG or Alexa Fluor-568 donkey anti-mouse IgG. The nucleus is stained with DAPI. SKOV-3 cells are analysed with LSM 510 META. The magnification used is ×200 (a). The percentage of apoptotic cells is calculated as follows: Apoptosis % = number of active caspase 3 positive cells/number of nuclei (b). C. Analysis of cleaved PARP by Western Blot. SKOV-3 cells are transfected with control or PAR-4 expressing plasmid (a) and with control or PAR-4 siRNA (b) and treated or not with 100 nM of taxol. After 48 hours, cell extracts are collected. Western Blot is performed and probed with anti-cleaved-PARP and anti-GAPDH antibodies. Bands of western blot are revealed by an ECL method, scanned and quantified by the Kodak 1D image analysis software. The cleaved-PARP band intensity is normalized to GAPDH and is expressed in percentage in function of control plasmid without taxol (c, d). D. Analysis of caspase3/7 activity with inhibition of PAR-4. SKOV-3 cells transfected with control or PAR-4 expressing plasmid are treated or not with 100 nM of taxol and 2.5 ug/ml of anti-PAR-4 antibodies (a) or anti-GRP78 antibodies (N20) (b) or IgG control for 24 hours. After 24 hours, caspase3/7 activity is examined by luminescent assay 24 post-treatment with antibodies. Quantitative results are presented in Figures a and b. E. Active caspase-3 staining in SKOV-3 cells treated or not with conditioned media. SKOV-3 cells are transfected with control or PAR-4 expressing plasmids. The culture supernatant is collected at 24, 48 h and 72 h post transfection (PT), and centrifuged at 14 000 rpm for 10 min. The supernatant is then aliquoted and frozen until use. Western blot is performed to evaluate the presence of secreted PAR-4 in medium (a). SKOV-3 cells are transfected with control or PAR-4 and GRP78 expressing plasmids. The efficiency of transfection is confirmed by western blot (data not shown). 24 hours after transfection, SKOV-3 cells are seeded in Lab-tek chamber. 24 hours after, SKOV-3 cells are treated or not with 100 nM of taxol and with 72 h PT conditioned medium for 24 hours. SKOV-3 cells are washed, fixed and then, incubated with anti-PAR-4 antibodies (R-334) and active caspase-3 antibodies. Then, cells are incubated for 1 hour with Alexa Fluor-488 donkey anti-rabbit IgG or Alexa Fluor-568 donkey anti-mouse IgG. The nucleus is stained with DAPI. SKOV-3 cells are analysed with LSM 510 META. The magnification used is ×200 (b). The percentage of apoptotic cells is calculated as follows: Apoptosis % = number of active caspase 3 positive cells/number of nuclei (c).
Figure Legend Snippet: Effect of PAR-4 levels on cell apoptosis A. Analysis of caspase 3/7 activity. SKOV-3 cells are transfected with control or PAR-4 expressing plasmid (a) and with control or PAR-4 siRNA (b) and treated or not with 100 nM of taxol. After 24 hours, luminescent assay is performed to measure caspase3/7 activity. Quantitative results are presented in Figures a and b. B. Apoptosis scoring of active caspase-3. SKOV-3 cells are transfected with control or PAR-4 expressing plasmid. 24 hours after, SKOV-3 cells are seeded and treated or not with 100 nM of taxol for 24 hours. SKOV-3 cells are washed, fixed and then, incubated with anti-PAR-4 antibodies (R-334) and active caspase-3 antibodies. Then, cells are incubated for 1 hour with Alexa Fluor-488 donkey anti-rabbit IgG or Alexa Fluor-568 donkey anti-mouse IgG. The nucleus is stained with DAPI. SKOV-3 cells are analysed with LSM 510 META. The magnification used is ×200 (a). The percentage of apoptotic cells is calculated as follows: Apoptosis % = number of active caspase 3 positive cells/number of nuclei (b). C. Analysis of cleaved PARP by Western Blot. SKOV-3 cells are transfected with control or PAR-4 expressing plasmid (a) and with control or PAR-4 siRNA (b) and treated or not with 100 nM of taxol. After 48 hours, cell extracts are collected. Western Blot is performed and probed with anti-cleaved-PARP and anti-GAPDH antibodies. Bands of western blot are revealed by an ECL method, scanned and quantified by the Kodak 1D image analysis software. The cleaved-PARP band intensity is normalized to GAPDH and is expressed in percentage in function of control plasmid without taxol (c, d). D. Analysis of caspase3/7 activity with inhibition of PAR-4. SKOV-3 cells transfected with control or PAR-4 expressing plasmid are treated or not with 100 nM of taxol and 2.5 ug/ml of anti-PAR-4 antibodies (a) or anti-GRP78 antibodies (N20) (b) or IgG control for 24 hours. After 24 hours, caspase3/7 activity is examined by luminescent assay 24 post-treatment with antibodies. Quantitative results are presented in Figures a and b. E. Active caspase-3 staining in SKOV-3 cells treated or not with conditioned media. SKOV-3 cells are transfected with control or PAR-4 expressing plasmids. The culture supernatant is collected at 24, 48 h and 72 h post transfection (PT), and centrifuged at 14 000 rpm for 10 min. The supernatant is then aliquoted and frozen until use. Western blot is performed to evaluate the presence of secreted PAR-4 in medium (a). SKOV-3 cells are transfected with control or PAR-4 and GRP78 expressing plasmids. The efficiency of transfection is confirmed by western blot (data not shown). 24 hours after transfection, SKOV-3 cells are seeded in Lab-tek chamber. 24 hours after, SKOV-3 cells are treated or not with 100 nM of taxol and with 72 h PT conditioned medium for 24 hours. SKOV-3 cells are washed, fixed and then, incubated with anti-PAR-4 antibodies (R-334) and active caspase-3 antibodies. Then, cells are incubated for 1 hour with Alexa Fluor-488 donkey anti-rabbit IgG or Alexa Fluor-568 donkey anti-mouse IgG. The nucleus is stained with DAPI. SKOV-3 cells are analysed with LSM 510 META. The magnification used is ×200 (b). The percentage of apoptotic cells is calculated as follows: Apoptosis % = number of active caspase 3 positive cells/number of nuclei (c).

Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation, Luminescence Assay, Incubation, Staining, Western Blot, Software, Inhibition

30) Product Images from "G-Protein-Coupled Estrogen Receptor Agonist Suppresses Airway Inflammation in a Mouse Model of Asthma through IL-10"

Article Title: G-Protein-Coupled Estrogen Receptor Agonist Suppresses Airway Inflammation in a Mouse Model of Asthma through IL-10

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123210

IL-10 deprivation abolished G-1-induced improvement of inflammatory cell accumulation in the lung. A : H E staining (original magnification: x40 and x400, upper panels) and PAS staining (x400, lower left panel) and MT staining (x400, lower right panel) of serial lung sections. B : Quantified data of inflammatory cell accumulation, histologically examined in H E–stained lung tissue, as described in Materials and Methods. C : Inflammatory cell accumulation in BAL fluid. Values are expressed as the mean ± SEM for G-1-treated mice (n = 6) and non-treated controls (n = 7).
Figure Legend Snippet: IL-10 deprivation abolished G-1-induced improvement of inflammatory cell accumulation in the lung. A : H E staining (original magnification: x40 and x400, upper panels) and PAS staining (x400, lower left panel) and MT staining (x400, lower right panel) of serial lung sections. B : Quantified data of inflammatory cell accumulation, histologically examined in H E–stained lung tissue, as described in Materials and Methods. C : Inflammatory cell accumulation in BAL fluid. Values are expressed as the mean ± SEM for G-1-treated mice (n = 6) and non-treated controls (n = 7).

Techniques Used: Staining, Mouse Assay

IL-10 depletion eradicated G-1-induced reduction of Th2 cytokines in BAL fluid and serum IgE. A , B : The levels of eotaxin, IL-4, IL-5, IL-10, IL-13, and IFN-γ in BAL fluid and total and OVA-specific IgE antibodies in serum of IL-10 KO mice were measured using ELISA. Values are expressed as the mean ± SEM for G-1-treated mice (n = 6) and non-treated controls (n = 7).
Figure Legend Snippet: IL-10 depletion eradicated G-1-induced reduction of Th2 cytokines in BAL fluid and serum IgE. A , B : The levels of eotaxin, IL-4, IL-5, IL-10, IL-13, and IFN-γ in BAL fluid and total and OVA-specific IgE antibodies in serum of IL-10 KO mice were measured using ELISA. Values are expressed as the mean ± SEM for G-1-treated mice (n = 6) and non-treated controls (n = 7).

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

G-1 treatment increased the frequency of IL-10 + CD4 + T cells and the secretion of IL-10 from splenocytes in C57BL/6 mice. A : Representative FACS data showing frequencies of IL-10 + CD4 + T cells and IL-10 + CD8 + T cells in splenocytes. B , C : Splenocytes were analyzed using a flow cytometer for the frequency of IL-10-producing CD4 + and CD8 + T cells. D : Splenocytes were analyzed using a flow cytometer for the frequency of Foxp3 + CD4 + T cells. E , F : Splenocytes were analyzed using a flow cytometer for the frequency of GPER-expressing cells and IL-10-producing GPER + CD4 + T cells. G : The levels of IL-10 in splenocyte culture supernatant were measured by means of ELISA. Values are expressed as the mean ± SEM. * P
Figure Legend Snippet: G-1 treatment increased the frequency of IL-10 + CD4 + T cells and the secretion of IL-10 from splenocytes in C57BL/6 mice. A : Representative FACS data showing frequencies of IL-10 + CD4 + T cells and IL-10 + CD8 + T cells in splenocytes. B , C : Splenocytes were analyzed using a flow cytometer for the frequency of IL-10-producing CD4 + and CD8 + T cells. D : Splenocytes were analyzed using a flow cytometer for the frequency of Foxp3 + CD4 + T cells. E , F : Splenocytes were analyzed using a flow cytometer for the frequency of GPER-expressing cells and IL-10-producing GPER + CD4 + T cells. G : The levels of IL-10 in splenocyte culture supernatant were measured by means of ELISA. Values are expressed as the mean ± SEM. * P

Techniques Used: Mouse Assay, FACS, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

31) Product Images from "miR-142 controls metabolic reprogramming that regulates dendritic cell activation"

Article Title: miR-142 controls metabolic reprogramming that regulates dendritic cell activation

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI123839

miR-142 modulates DC’s TLR responses in vitro. ( A ) CD11c + DCs were quantified from spleens of WT or miR-142 –/– mice ( n = 30, mean ± SEM). Splenic WT or miR-142 –/– DCs were treated with LPS or diluent control and ( B ) analyzed for expression of MHC II, CD80, CD86, CD40, annexin V (mean ± SEM) (data are combined from 4 independent experiments), and ( C ) cytokine levels by ELISA (mean ± SEM) (data are combined from 3 independent experiments). ( D ) Splenic WT or miR-142 –/– DCs were treated with TLR ligands LPS, Flagellin, Pam3CSK4, and Poly(I:C) or diluent control and analyzed for expression of MHC II, CD80, CD86, and CD40 (mean ± SEM) (data are combined from 4 independent experiments) and ( E ) cytokine levels by ELISA (mean ± SEM) (data are combined from 3 independent experiments). Comparisons between 2 groups were calculated using paired Student’s t test (2 tailed), while comparisons between 2 groups at multiple time points were calculated utilizing multiple t tests (Holm-Šidák method). * P
Figure Legend Snippet: miR-142 modulates DC’s TLR responses in vitro. ( A ) CD11c + DCs were quantified from spleens of WT or miR-142 –/– mice ( n = 30, mean ± SEM). Splenic WT or miR-142 –/– DCs were treated with LPS or diluent control and ( B ) analyzed for expression of MHC II, CD80, CD86, CD40, annexin V (mean ± SEM) (data are combined from 4 independent experiments), and ( C ) cytokine levels by ELISA (mean ± SEM) (data are combined from 3 independent experiments). ( D ) Splenic WT or miR-142 –/– DCs were treated with TLR ligands LPS, Flagellin, Pam3CSK4, and Poly(I:C) or diluent control and analyzed for expression of MHC II, CD80, CD86, and CD40 (mean ± SEM) (data are combined from 4 independent experiments) and ( E ) cytokine levels by ELISA (mean ± SEM) (data are combined from 3 independent experiments). Comparisons between 2 groups were calculated using paired Student’s t test (2 tailed), while comparisons between 2 groups at multiple time points were calculated utilizing multiple t tests (Holm-Šidák method). * P

Techniques Used: In Vitro, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay

32) Product Images from "Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors"

Article Title: Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006740

Ruxolitinib does not inhibit normal TCR function and signaling that is independent of HIV-1 infection. (A) Mean CD3 zeta and SLP76 phosphorylation (MFI in CD4 cells) as quantified by flow cytometry in CD4 + cells isolated from HIV negative donors and stimulated with anti-CD3/CD28 in the presence of increasing concentrations of Ruxoltinib versus DMSO treated control cells (n = 3). Statistical significance was determined by an upaired t-test corrected for multiple comparisons using the Holm-Sidak method. (B) Mean cytokine production (% of IL-2 + , TNF-α + and IFN-γ + triple positive cells) in CD3+CD8- cells or CD3+CD8+ cells as measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 6 hr with aCD3/CD28, Brefeldin A (5 μg/ml) and increasing concentrations of Ruxoltinib versus DMSO treated cells (n = 3). Statistical significance for (B) determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p
Figure Legend Snippet: Ruxolitinib does not inhibit normal TCR function and signaling that is independent of HIV-1 infection. (A) Mean CD3 zeta and SLP76 phosphorylation (MFI in CD4 cells) as quantified by flow cytometry in CD4 + cells isolated from HIV negative donors and stimulated with anti-CD3/CD28 in the presence of increasing concentrations of Ruxoltinib versus DMSO treated control cells (n = 3). Statistical significance was determined by an upaired t-test corrected for multiple comparisons using the Holm-Sidak method. (B) Mean cytokine production (% of IL-2 + , TNF-α + and IFN-γ + triple positive cells) in CD3+CD8- cells or CD3+CD8+ cells as measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 6 hr with aCD3/CD28, Brefeldin A (5 μg/ml) and increasing concentrations of Ruxoltinib versus DMSO treated cells (n = 3). Statistical significance for (B) determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p

Techniques Used: Infection, Flow Cytometry, Cytometry, Isolation

Jak inhibitors block HIV-1 replication and production ex vivo and in vitro . Viral production was measured by ELISA p24 in cell-free supernatants of enriched CD4 + T cells isolated from 5 viremic donors and stimulated for 6 days with anti-CD3/28 in the presence of increasing concentrations of Jak inhibitors without (A) or with (B) ART. Viral production was measured by ELISA p24 in cell-free supernatants of in vitro infected CD4 + cells after 6 days culture in the presence of increasing concentrations of Jak inhibitors (C) and by the frequency of HIV-GFP+ expressing CD4+ cells after 3 day culture (D). To account for inter-patient variability in baseline values, results are reported as the fold change versus DMSO controls. 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p
Figure Legend Snippet: Jak inhibitors block HIV-1 replication and production ex vivo and in vitro . Viral production was measured by ELISA p24 in cell-free supernatants of enriched CD4 + T cells isolated from 5 viremic donors and stimulated for 6 days with anti-CD3/28 in the presence of increasing concentrations of Jak inhibitors without (A) or with (B) ART. Viral production was measured by ELISA p24 in cell-free supernatants of in vitro infected CD4 + cells after 6 days culture in the presence of increasing concentrations of Jak inhibitors (C) and by the frequency of HIV-GFP+ expressing CD4+ cells after 3 day culture (D). To account for inter-patient variability in baseline values, results are reported as the fold change versus DMSO controls. 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p

Techniques Used: Blocking Assay, Ex Vivo, In Vitro, Enzyme-linked Immunosorbent Assay, Isolation, Infection, Expressing

Ruxolitinib inhibits bystander infection. Uninfected CD4 + T cells were incubated with or without cell trace violet (CTV) dye. Cells with CTV dye were stimulated with CD3/CD28 and various concentrations of ruxolitinib or DMSO for 3 days (A, top). Cells without CTV dye were incubated with CD3/CD28 for 3 days followed by a 2 hours spinoculation with a replication competent eGFP Nl4-3 X4 HIV-1 (A, bottom). After spinocualtion on Day 3, both cultures (traced and untraced) were co-incubated for two days in the absence of ruxolitinib. Representative dot plots for bystander infection quantification are demonstrated in panel B. Ruxolitinib inhibits bystander infection (GFP and CTV double positive) of uninfected bystander cells (CTV + ) in a dose dependent manner (B and C, n = 3). Ruxolitinib blocks proliferation (CTV-lo) of bystander cells in a dose dependent manner with all concentrations tested (D). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent mean with S.E.M (C) or mean with standard deviation (D) and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test (C; ****p
Figure Legend Snippet: Ruxolitinib inhibits bystander infection. Uninfected CD4 + T cells were incubated with or without cell trace violet (CTV) dye. Cells with CTV dye were stimulated with CD3/CD28 and various concentrations of ruxolitinib or DMSO for 3 days (A, top). Cells without CTV dye were incubated with CD3/CD28 for 3 days followed by a 2 hours spinoculation with a replication competent eGFP Nl4-3 X4 HIV-1 (A, bottom). After spinocualtion on Day 3, both cultures (traced and untraced) were co-incubated for two days in the absence of ruxolitinib. Representative dot plots for bystander infection quantification are demonstrated in panel B. Ruxolitinib inhibits bystander infection (GFP and CTV double positive) of uninfected bystander cells (CTV + ) in a dose dependent manner (B and C, n = 3). Ruxolitinib blocks proliferation (CTV-lo) of bystander cells in a dose dependent manner with all concentrations tested (D). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent mean with S.E.M (C) or mean with standard deviation (D) and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test (C; ****p

Techniques Used: Infection, Incubation, Standard Deviation

Jak inhibitors reduce frequency of cells harboring integrated viral DNA and IL-15-induced reactivation of latent HIV-1 in CD4 T cells. CD4 T cells were isolated from viremic donors and incubated with CD3/CD28 plus 0.01, 0.1, 1.0 or 10 μM of Jak inhibitors with or without EC 99 of ART (180 nM zidovudine, 100 nM efavirenz, 200 nM Raltegravir) (A and B). After six days, integrated viral DNA was quantified using ultra sensitive Alu PCR versus DMSO controls (n = 5). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p
Figure Legend Snippet: Jak inhibitors reduce frequency of cells harboring integrated viral DNA and IL-15-induced reactivation of latent HIV-1 in CD4 T cells. CD4 T cells were isolated from viremic donors and incubated with CD3/CD28 plus 0.01, 0.1, 1.0 or 10 μM of Jak inhibitors with or without EC 99 of ART (180 nM zidovudine, 100 nM efavirenz, 200 nM Raltegravir) (A and B). After six days, integrated viral DNA was quantified using ultra sensitive Alu PCR versus DMSO controls (n = 5). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p

Techniques Used: Isolation, Incubation, Polymerase Chain Reaction

Pharmacokinetic simulation for 10 mg and 20 mg bid dosing of ruxolitinib demonstrates that anti-HIV effects occur at physiologically relevant concentrations observed in humans. Simulation of in vivo pharmacokinetics of 10 mg (A) or 20 mg (B) bid ruxolitinib demonstrated that all pro-HIV events that were inhibited by ruxolitinib in vitro occur at or below concentrations within the steady state plasma concentrations observed in vivo for 10 mg bid (A), and 20 mg bid (B). Dotted lines denote IC 50 at which ruxolitinib confers inhibition in vitro , and notations of A-D denote: CD3 zeta and pSLP76, A; inhibition of Bcl-2 activation, B; inhibition of maintenance and expansion of the T cell reservoir, and antiviral potency against chronic and acute infection, C; inhibition of proliferation/activation (CD38/HLADR, PD1), down regulation of CCR5, inhibition of pSTAT5 by IL-2, IL-7, IL-15, inhibition of bystander infection, D.
Figure Legend Snippet: Pharmacokinetic simulation for 10 mg and 20 mg bid dosing of ruxolitinib demonstrates that anti-HIV effects occur at physiologically relevant concentrations observed in humans. Simulation of in vivo pharmacokinetics of 10 mg (A) or 20 mg (B) bid ruxolitinib demonstrated that all pro-HIV events that were inhibited by ruxolitinib in vitro occur at or below concentrations within the steady state plasma concentrations observed in vivo for 10 mg bid (A), and 20 mg bid (B). Dotted lines denote IC 50 at which ruxolitinib confers inhibition in vitro , and notations of A-D denote: CD3 zeta and pSLP76, A; inhibition of Bcl-2 activation, B; inhibition of maintenance and expansion of the T cell reservoir, and antiviral potency against chronic and acute infection, C; inhibition of proliferation/activation (CD38/HLADR, PD1), down regulation of CCR5, inhibition of pSTAT5 by IL-2, IL-7, IL-15, inhibition of bystander infection, D.

Techniques Used: In Vivo, In Vitro, Inhibition, Activation Assay, Infection

33) Product Images from "The arthritis-associated HLA-B*27:05 allele forms more cell surface B27 dimer and free heavy chain ligands for KIR3DL2 than HLA-B*27:09"

Article Title: The arthritis-associated HLA-B*27:05 allele forms more cell surface B27 dimer and free heavy chain ligands for KIR3DL2 than HLA-B*27:09

Journal: Rheumatology (Oxford, England)

doi: 10.1093/rheumatology/ket219

Increased expression of KIR3DL2 by leucocytes from HLA-B*27:05+ patients compared with HLA-B27− and HLA-B*27:05+ and HLA-B*27:09+ healthy controls (HCs). ( A ) Left hand panel: percentage of NK cells expressing KIR3DL2 in HLA-B*27:05 + SpA patients, HLA-B*27:05+ HC, HLA-B*27:09 HC and HLA-B27 negative HC. Percentages of NK cells expressing KIR3DL2 were 35.6 ± 13.6 (mean ± 1 s.d. ), 20.3 ± 6.3 ( P = 0.0004), 17.9 ± 11.3 ( P = 0.0007) and 22.3 ± 8.5 ( P = 0.007) for each of the respective groups. Right hand panel: level of expression of KIR3DL2 (ABC units) by SpA patient NK cells, HLA-B*27:05+, HLA-B*27:09+ and HLA-B27− HC. HLA-B*27:05+ SpA patients expressed 1571 ± 631.9 ABC units compared with 1038 ± 235.7 units ( P = 0.003) for HLA-B*27:05 HC, 1049 ± 331.5 ( P = 0.007) for HLA-B*27:09+ HC and 961 ± 318 units ( P = 0.003) for HLA-B27 negative HC. ( B ) Left hand panel: percentages of CD4 T cells expressing KIR3DL2 in HLA-B*27:05 + SpA patients, HLA-B*27:05+, HLA-B*27:09 + and HLA-B27 negative HC. A mean of 1.8 ± 1.3 SpA CD4 T cells, 0.8 ± 0.4 HLA-B*27:05 HC ( P = 0.01), 0.9 ± 0.3 B*27:09 HC ( P = 0.009) and 1.0 ± 0.7 ( P = 0.04) B27 negative HC expressed KIR3DL2. Right hand panel: SpA patient CD4 T cells expressed 1232 ± 513.8 ABC units of KIR3DL2, compared with 878.3 ± 199 HLA-B*27:05 HC ( P = 0.011), 859.3 ± 288.7 HLA-B*27:09 HC ( P = 0.016) and 847 ± 321 B27 negative HC ( P = 0.030).
Figure Legend Snippet: Increased expression of KIR3DL2 by leucocytes from HLA-B*27:05+ patients compared with HLA-B27− and HLA-B*27:05+ and HLA-B*27:09+ healthy controls (HCs). ( A ) Left hand panel: percentage of NK cells expressing KIR3DL2 in HLA-B*27:05 + SpA patients, HLA-B*27:05+ HC, HLA-B*27:09 HC and HLA-B27 negative HC. Percentages of NK cells expressing KIR3DL2 were 35.6 ± 13.6 (mean ± 1 s.d. ), 20.3 ± 6.3 ( P = 0.0004), 17.9 ± 11.3 ( P = 0.0007) and 22.3 ± 8.5 ( P = 0.007) for each of the respective groups. Right hand panel: level of expression of KIR3DL2 (ABC units) by SpA patient NK cells, HLA-B*27:05+, HLA-B*27:09+ and HLA-B27− HC. HLA-B*27:05+ SpA patients expressed 1571 ± 631.9 ABC units compared with 1038 ± 235.7 units ( P = 0.003) for HLA-B*27:05 HC, 1049 ± 331.5 ( P = 0.007) for HLA-B*27:09+ HC and 961 ± 318 units ( P = 0.003) for HLA-B27 negative HC. ( B ) Left hand panel: percentages of CD4 T cells expressing KIR3DL2 in HLA-B*27:05 + SpA patients, HLA-B*27:05+, HLA-B*27:09 + and HLA-B27 negative HC. A mean of 1.8 ± 1.3 SpA CD4 T cells, 0.8 ± 0.4 HLA-B*27:05 HC ( P = 0.01), 0.9 ± 0.3 B*27:09 HC ( P = 0.009) and 1.0 ± 0.7 ( P = 0.04) B27 negative HC expressed KIR3DL2. Right hand panel: SpA patient CD4 T cells expressed 1232 ± 513.8 ABC units of KIR3DL2, compared with 878.3 ± 199 HLA-B*27:05 HC ( P = 0.011), 859.3 ± 288.7 HLA-B*27:09 HC ( P = 0.016) and 847 ± 321 B27 negative HC ( P = 0.030).

Techniques Used: Expressing

Similar binding of HLA-B*27:05 and HLA-B*27:09 dimers to KIR3DL1, KIR3DL2 and LILRB2. HLA-B*27:05 and HLA-B*27:09 heterodimers bind differently to KIR3DL1. ( A ) Representative FACS staining of LILRB2-transduced Baf3 cells with HLAB*27:05 (B*27:05 2 ) and HLA-B*27:09 (B*27:09 2 ) dimer tetramers. Cells were stained with extravidin PE (EX PE) as a negative control stain. ( B ) Representative FACS staining of KIR3DL1- and KIR3DL2-transduced Baf3 cells with HLA-B*27:05 (B*27:05 2 ) and HLA-B*27:09 (B*27:09 2 ) heavy chain dimer tetramers. Representative staining of KIR3DL2-transduced Baf3 cells with HLA-B*27:05 (B*27:05) and HLA-B*27:09 heterodimer (B*27:09) tetramers. Staining with HLA-B*27:05 (B*27:05 2 ) heavy chain dimer tetramers (B27 2 ) is shown for comparison. Cells were stained with extravidin PE (EX PE) as a negative control stain. ( C ) Representative FACS staining of KIR3DL1- and LILRB1-transduced Baf3 cells with HLA-B*27:05 (B*27:05) and HLA-B*27:09 (B*27:09) heterodimer tetramers formed with the FluNP epitope. Cells were stained with extravidin PE (EX PE) as a negative control stain. ( D ) Representative FACS staining of KIR3DL1- and LILRB1-transduced Baf3 cells with HLA-B*27:05 (B*27:05) and HLA-B*27:09 (B*27:09) heterodimer tetramers formed with the HIV GAG epitope. Cells were stained with extravidin PE (EX PE) as a negative control stain.
Figure Legend Snippet: Similar binding of HLA-B*27:05 and HLA-B*27:09 dimers to KIR3DL1, KIR3DL2 and LILRB2. HLA-B*27:05 and HLA-B*27:09 heterodimers bind differently to KIR3DL1. ( A ) Representative FACS staining of LILRB2-transduced Baf3 cells with HLAB*27:05 (B*27:05 2 ) and HLA-B*27:09 (B*27:09 2 ) dimer tetramers. Cells were stained with extravidin PE (EX PE) as a negative control stain. ( B ) Representative FACS staining of KIR3DL1- and KIR3DL2-transduced Baf3 cells with HLA-B*27:05 (B*27:05 2 ) and HLA-B*27:09 (B*27:09 2 ) heavy chain dimer tetramers. Representative staining of KIR3DL2-transduced Baf3 cells with HLA-B*27:05 (B*27:05) and HLA-B*27:09 heterodimer (B*27:09) tetramers. Staining with HLA-B*27:05 (B*27:05 2 ) heavy chain dimer tetramers (B27 2 ) is shown for comparison. Cells were stained with extravidin PE (EX PE) as a negative control stain. ( C ) Representative FACS staining of KIR3DL1- and LILRB1-transduced Baf3 cells with HLA-B*27:05 (B*27:05) and HLA-B*27:09 (B*27:09) heterodimer tetramers formed with the FluNP epitope. Cells were stained with extravidin PE (EX PE) as a negative control stain. ( D ) Representative FACS staining of KIR3DL1- and LILRB1-transduced Baf3 cells with HLA-B*27:05 (B*27:05) and HLA-B*27:09 (B*27:09) heterodimer tetramers formed with the HIV GAG epitope. Cells were stained with extravidin PE (EX PE) as a negative control stain.

Techniques Used: Binding Assay, FACS, Staining, Negative Control

KIR3DL2 binds more strongly to cell surface HLA-B*27:05 than HLA-B*27:09. ( A ) IL-2 secretion by KIR3DL2CD3ε-reporter Jurkat T cells stimulated with ( A ) HLA-B*27:05 and HLA-B*27:09 transduced 221 cells. Effect of HC10, HD6, ME1 and W632 MAbs on IL-2 secretion by KIR3DL2CD3ε-transduced Jurkat T cells is also shown. Representative of three independent experiments. ( B ) Left hand panels: proportions of viable (Annexin V, Live-Dead negative) KIR3DL2 + NK cells after 6 day stimulation with parental 221 cells or 221B*27:05 and 221B*27:09 cells. Right hand panels: pproportions of viable (Annexin V, Live-Dead negative) KIR3DL2 + NK cells after 6 day stimulation with 221B*27:05 cells and anti-KIR3DL2 (DX31) or HC10 and W632 antibodies or isotype control antibody (IgG2a). Representative FACS stains from one of three independent experiments with an NK cell line from a healthy B27 control. ( C ) Proportions of viable KIR3DL2 + NK cells after 6 day stimulation with 221, 221B*27:05 or 221B*27:09 cells or 221B*27:05 cells with anti-KIR3DL2 (DX31), HC10, W632 or isotype control (IgG2a) antibodies. Mean proportions of surviving cells ± 1 s.d. from three independent experiments. * P
Figure Legend Snippet: KIR3DL2 binds more strongly to cell surface HLA-B*27:05 than HLA-B*27:09. ( A ) IL-2 secretion by KIR3DL2CD3ε-reporter Jurkat T cells stimulated with ( A ) HLA-B*27:05 and HLA-B*27:09 transduced 221 cells. Effect of HC10, HD6, ME1 and W632 MAbs on IL-2 secretion by KIR3DL2CD3ε-transduced Jurkat T cells is also shown. Representative of three independent experiments. ( B ) Left hand panels: proportions of viable (Annexin V, Live-Dead negative) KIR3DL2 + NK cells after 6 day stimulation with parental 221 cells or 221B*27:05 and 221B*27:09 cells. Right hand panels: pproportions of viable (Annexin V, Live-Dead negative) KIR3DL2 + NK cells after 6 day stimulation with 221B*27:05 cells and anti-KIR3DL2 (DX31) or HC10 and W632 antibodies or isotype control antibody (IgG2a). Representative FACS stains from one of three independent experiments with an NK cell line from a healthy B27 control. ( C ) Proportions of viable KIR3DL2 + NK cells after 6 day stimulation with 221, 221B*27:05 or 221B*27:09 cells or 221B*27:05 cells with anti-KIR3DL2 (DX31), HC10, W632 or isotype control (IgG2a) antibodies. Mean proportions of surviving cells ± 1 s.d. from three independent experiments. * P

Techniques Used: FACS

34) Product Images from "TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation"

Article Title: TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02612

TACI deficiency extends formation and resolution kinetics of T FH and GC response. TACI -/- and C57BL/6 mice were infected (i.p.) with 1 × 10 6 P. yoelii (Py 17XNL strain) parasites. (A) Representative dot plots depict the percentage of splenic PD-1 high CXCR5 high (T FH ) cell on CD44+CD4+ pre-gated T cells at day 15 post-infection. (B) Formation and resolution kinetics of T FH cells presented as number of T FH cells per spleen. (C) Representative dot plots depict the percentage of and GL-7 hig hFAS high (GC B cells) on B220 + pre-gated B cells at day 15 post-infection. (D) Formation and resolution ki“netics of GC B cells presented as number of GC B cells per spleen. Total splenic B cell (E) Day 10 post-infection ICOSL expression levels were measured on B220 + GL-7 high FAS high gated splenic GC B cells. Representative histograms as well as frequencies of ICOSL expressing cells and ICOSL MFI for each mouse strain are shown. Unpaired Student's t -test was used for statistical evaluation. Results are expressed as mean ± SEM ( n = 5) from one representative experiment out of three with similar results. * p
Figure Legend Snippet: TACI deficiency extends formation and resolution kinetics of T FH and GC response. TACI -/- and C57BL/6 mice were infected (i.p.) with 1 × 10 6 P. yoelii (Py 17XNL strain) parasites. (A) Representative dot plots depict the percentage of splenic PD-1 high CXCR5 high (T FH ) cell on CD44+CD4+ pre-gated T cells at day 15 post-infection. (B) Formation and resolution kinetics of T FH cells presented as number of T FH cells per spleen. (C) Representative dot plots depict the percentage of and GL-7 hig hFAS high (GC B cells) on B220 + pre-gated B cells at day 15 post-infection. (D) Formation and resolution ki“netics of GC B cells presented as number of GC B cells per spleen. Total splenic B cell (E) Day 10 post-infection ICOSL expression levels were measured on B220 + GL-7 high FAS high gated splenic GC B cells. Representative histograms as well as frequencies of ICOSL expressing cells and ICOSL MFI for each mouse strain are shown. Unpaired Student's t -test was used for statistical evaluation. Results are expressed as mean ± SEM ( n = 5) from one representative experiment out of three with similar results. * p

Techniques Used: Mouse Assay, Infection, Expressing

PC output from GC is limited in TACI -/- mice. TACI -/- and C57BL/6 mice were infected (i.p.) with 1 × 10 6 P. yoelii (Py 17XNL) parasites. Splenic GC and PC were examined by microscopy on indicated days after infection. (A) Histologic sections of spleens were stained with anti-B220 (green) and anti-GL-7 (red) antibodies to visualize B-cell follicles (green), GC (B220 + GL-7 + , yellow), and PC (B220 low GL-7 + , orange). T-cell independent PC are located in the marginal zone and T-cell dependent PC in red pulp of the spleen, adjacent to the “Bridging channels.” T cell zones were defined as central location where tightly packed follicles are distributed around and the activated T cells (B220 − GL-7 + , red) are located. Arrows indicate the plasmablast pathway to extrafollicular areas through the “Bridging channels.” (B) PC output was calculated by ratio of PC foci to GCs in half spleen. Each dot represents one mouse. Magnification is 10x. * p
Figure Legend Snippet: PC output from GC is limited in TACI -/- mice. TACI -/- and C57BL/6 mice were infected (i.p.) with 1 × 10 6 P. yoelii (Py 17XNL) parasites. Splenic GC and PC were examined by microscopy on indicated days after infection. (A) Histologic sections of spleens were stained with anti-B220 (green) and anti-GL-7 (red) antibodies to visualize B-cell follicles (green), GC (B220 + GL-7 + , yellow), and PC (B220 low GL-7 + , orange). T-cell independent PC are located in the marginal zone and T-cell dependent PC in red pulp of the spleen, adjacent to the “Bridging channels.” T cell zones were defined as central location where tightly packed follicles are distributed around and the activated T cells (B220 − GL-7 + , red) are located. Arrows indicate the plasmablast pathway to extrafollicular areas through the “Bridging channels.” (B) PC output was calculated by ratio of PC foci to GCs in half spleen. Each dot represents one mouse. Magnification is 10x. * p

Techniques Used: Mouse Assay, Infection, Microscopy, Staining

35) Product Images from "A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells"

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

Journal: Journal of Virology

doi: 10.1128/JVI.01956-16

Recruitment of GC B cells by CXCL13. BALB/c mice ( n = 3) were infected via i.m. injection of 1 × 10 6 FFU of different rRABVs, and the spleens and draining LNs were harvested at 7 and 14 dpi. Single-cell suspensions were prepared, stained with antibodies against GC B cells and GC B cell activation markers, and analyzed via flow cytometry. (A and B) Representative gating strategies for the detection of GC B cells (A) and representative flow cytometric plots of GC B cells (B) are shown. (C and D) The results of a detailed analysis for activated GC B cells (B220 + CD95 + GL7 + ) at 7 and 14 dpi are presented for the spleen (C) and the draining LNs (D). Data are presented as the means ± SEM ( n = 3). Asterisks indicate significant differences between the indicated experimental groups.
Figure Legend Snippet: Recruitment of GC B cells by CXCL13. BALB/c mice ( n = 3) were infected via i.m. injection of 1 × 10 6 FFU of different rRABVs, and the spleens and draining LNs were harvested at 7 and 14 dpi. Single-cell suspensions were prepared, stained with antibodies against GC B cells and GC B cell activation markers, and analyzed via flow cytometry. (A and B) Representative gating strategies for the detection of GC B cells (A) and representative flow cytometric plots of GC B cells (B) are shown. (C and D) The results of a detailed analysis for activated GC B cells (B220 + CD95 + GL7 + ) at 7 and 14 dpi are presented for the spleen (C) and the draining LNs (D). Data are presented as the means ± SEM ( n = 3). Asterisks indicate significant differences between the indicated experimental groups.

Techniques Used: Mouse Assay, Infection, Injection, Staining, Activation Assay, Flow Cytometry, Cytometry

36) Product Images from "Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma"

Article Title: Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma

Journal: International Journal of Implant Dentistry

doi: 10.1186/s40729-018-0134-6

Effects of storage time on the coagulation pathway and platelets. Prothrombin time ( a ) and clot formation time ( b ) of citrated whole-blood samples stored for up to 6 days were examined simultaneously. Platelets’ responsiveness to Ca 2+ was assessed by upregulation of CD62P ( c ) and CD63 ( d ) in CD41 + platelets
Figure Legend Snippet: Effects of storage time on the coagulation pathway and platelets. Prothrombin time ( a ) and clot formation time ( b ) of citrated whole-blood samples stored for up to 6 days were examined simultaneously. Platelets’ responsiveness to Ca 2+ was assessed by upregulation of CD62P ( c ) and CD63 ( d ) in CD41 + platelets

Techniques Used: Coagulation

Immunofluorescent (IF) evaluation of changes in surface marker expression—CD41 ( a ), CD62P ( b ), and CD63 ( c )—in Ca 2+ -stimulated platelets. PLT platelets. Washed platelets were treated with 0.1% CaCl 2 in culture dishes and subjected to IF staining. Similar results were obtained from samples of three other donors
Figure Legend Snippet: Immunofluorescent (IF) evaluation of changes in surface marker expression—CD41 ( a ), CD62P ( b ), and CD63 ( c )—in Ca 2+ -stimulated platelets. PLT platelets. Washed platelets were treated with 0.1% CaCl 2 in culture dishes and subjected to IF staining. Similar results were obtained from samples of three other donors

Techniques Used: Marker, Expressing, Staining

37) Product Images from "Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain"

Article Title: Re-evaluation of IL-10 signaling reveals novel insights on the contribution of the intracellular domain of the IL-10R2 chain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0186317

IL-10R2 mediated signaling via Tyk2 plays a limited role in IL-10 activity. Bone marrow-derived macrophages, dendritic cells and mast cells from wild-type, IL-10R1 -/- , IL-10R2 -/- and Tyk2 -/- mice were tested for their response to IL-10. Macrophages and dendritic cells from wild-type and IL-10R -/- mice were pre-treated with IL-10 and subsequently stimulated with 100 ng/ml LPS. The percentage of inhibition of TNF-α expression of macrophages and dendritic cells was determined after overnight incubation (A and B, respectively) ( n = 3, error bars indicate standard error). Similarly, macrophages and dendritic cells from Tyk2 -/- mice were tested for their response to IL-10 (D and E, respectively) ( n = 4, error bars indicate standard error). Mast cells from wild-type and transgenic mice were cultured for 48 hours in the presence of IL-10 and cell viability was determined (C and F) ( n = 3 for IL-10R -/- mice and n = 4 for Tyk2 -/- mice, error bars indicate standard error). Asterisk(s) indicate significant differences as determined by a Welch’s t -test (* P
Figure Legend Snippet: IL-10R2 mediated signaling via Tyk2 plays a limited role in IL-10 activity. Bone marrow-derived macrophages, dendritic cells and mast cells from wild-type, IL-10R1 -/- , IL-10R2 -/- and Tyk2 -/- mice were tested for their response to IL-10. Macrophages and dendritic cells from wild-type and IL-10R -/- mice were pre-treated with IL-10 and subsequently stimulated with 100 ng/ml LPS. The percentage of inhibition of TNF-α expression of macrophages and dendritic cells was determined after overnight incubation (A and B, respectively) ( n = 3, error bars indicate standard error). Similarly, macrophages and dendritic cells from Tyk2 -/- mice were tested for their response to IL-10 (D and E, respectively) ( n = 4, error bars indicate standard error). Mast cells from wild-type and transgenic mice were cultured for 48 hours in the presence of IL-10 and cell viability was determined (C and F) ( n = 3 for IL-10R -/- mice and n = 4 for Tyk2 -/- mice, error bars indicate standard error). Asterisk(s) indicate significant differences as determined by a Welch’s t -test (* P

Techniques Used: Activity Assay, Derivative Assay, Mouse Assay, Inhibition, Expressing, Incubation, Transgenic Assay, Cell Culture

The intracellular domain of IL-10R2 mediates conformational changes in IL-10R1. CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2 Δ230–330 , IL-10R2 Δ1–190 or an empty vector (mcs) and cultured for 24 hours. Cells were surface stained with the 1B1.3a anti-mouse IL-10R1 monoclonal antibody and analysed by flow cytometry (A). Mean fluorescent intensity for IL-10R1 binding is plotted in a dose-dependent manner ( n = 6, error bars represent standard error). IL-10 was cross-linked to the surface of transfected cells and surface-bound IL-10 was detected by flow cytometry (B). Mean fluorescent intensity for IL-10 binding is plotted ( n = 3, error bars represent standard error). Asterisk(s) indicate significant differences as determined by a Welch’s t -test (* P
Figure Legend Snippet: The intracellular domain of IL-10R2 mediates conformational changes in IL-10R1. CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2 Δ230–330 , IL-10R2 Δ1–190 or an empty vector (mcs) and cultured for 24 hours. Cells were surface stained with the 1B1.3a anti-mouse IL-10R1 monoclonal antibody and analysed by flow cytometry (A). Mean fluorescent intensity for IL-10R1 binding is plotted in a dose-dependent manner ( n = 6, error bars represent standard error). IL-10 was cross-linked to the surface of transfected cells and surface-bound IL-10 was detected by flow cytometry (B). Mean fluorescent intensity for IL-10 binding is plotted ( n = 3, error bars represent standard error). Asterisk(s) indicate significant differences as determined by a Welch’s t -test (* P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Cell Culture, Staining, Flow Cytometry, Cytometry, Binding Assay

The extracellular domain of IL-10R2 is not sufficient to maintain IL-10 activity. CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2 Δ230–330 (extracellular domain), IL-10R2 Δ1–190 (intracellular domain) or an empty vector (mcs) and cultured for 24 hours. Surface expression of IL-10R1 or IL-10R2 was analysed by flow cytometry and mean fluorescent intensity is plotted ( n = 6, error bars represent standard error) (A). Phosphorylation of tyrosine 705 (Y705) of STAT3 by IL-10 (100 ng/ml) in CHO-K1 cells upon transient transfection with IL-10 receptors was analysed by western blot (B).
Figure Legend Snippet: The extracellular domain of IL-10R2 is not sufficient to maintain IL-10 activity. CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2 Δ230–330 (extracellular domain), IL-10R2 Δ1–190 (intracellular domain) or an empty vector (mcs) and cultured for 24 hours. Surface expression of IL-10R1 or IL-10R2 was analysed by flow cytometry and mean fluorescent intensity is plotted ( n = 6, error bars represent standard error) (A). Phosphorylation of tyrosine 705 (Y705) of STAT3 by IL-10 (100 ng/ml) in CHO-K1 cells upon transient transfection with IL-10 receptors was analysed by western blot (B).

Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Flow Cytometry, Cytometry, Western Blot

38) Product Images from "Indoleamine-2,3-dioxygenase and Interleukin-6 associated with tumor response to neoadjuvant chemotherapy in breast cancer"

Article Title: Indoleamine-2,3-dioxygenase and Interleukin-6 associated with tumor response to neoadjuvant chemotherapy in breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.22253

The association between IDO, IL-6 expression and response to neoadjuvant chemotherapy (A) The association between IDO and CR+PR. (B) The association between IL-6 and CR+PR. (C) The association between IDO and pCR. (D) The association between IL-6 and pCR.
Figure Legend Snippet: The association between IDO, IL-6 expression and response to neoadjuvant chemotherapy (A) The association between IDO and CR+PR. (B) The association between IL-6 and CR+PR. (C) The association between IDO and pCR. (D) The association between IL-6 and pCR.

Techniques Used: Expressing, Polymerase Chain Reaction

The association between IDO, IL-6 expression and plasma IL-6 level (A) The expression of IDO and IL-6 in tumor tissues. (B) Patients with high IDO expression more frequently had higher IL-6 high expression. (C) By Spearman's rank correlation test, it was found that the sum score of IDO correlated with the sum score of IL-6 ( r 2 =0.665, P
Figure Legend Snippet: The association between IDO, IL-6 expression and plasma IL-6 level (A) The expression of IDO and IL-6 in tumor tissues. (B) Patients with high IDO expression more frequently had higher IL-6 high expression. (C) By Spearman's rank correlation test, it was found that the sum score of IDO correlated with the sum score of IL-6 ( r 2 =0.665, P

Techniques Used: Expressing

The survival curves for breast cancer patients treated by neoadjuvant chemotherapy The survival curves for PFS and OS according to different clinical T stage, pCR, IDO expression, IL-6 expression, IDO/IL-6 co-expression were shown.
Figure Legend Snippet: The survival curves for breast cancer patients treated by neoadjuvant chemotherapy The survival curves for PFS and OS according to different clinical T stage, pCR, IDO expression, IL-6 expression, IDO/IL-6 co-expression were shown.

Techniques Used: Polymerase Chain Reaction, Expressing

39) Product Images from "Distinct In Vitro T-Helper 17 Differentiation Capacity of Peripheral Naive T Cells in Rheumatoid and Psoriatic Arthritis"

Article Title: Distinct In Vitro T-Helper 17 Differentiation Capacity of Peripheral Naive T Cells in Rheumatoid and Psoriatic Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00606

RORC and TBX21 expression during T-helper 17 (Th17) differentiation. Naive CD4 + T cells were differentiated toward Th17-lane. Samples were treated with CD3/CD28/CAB (stim) or with stim + anti-IL4 antibody and the following four cytokine combinations to promote naive CD4 + T cell differentiation: transforming grow factor beta (TGF β ) + IL6, TGF β + IL6 + IL1 β , IL1 β + IL23, and IL1 β + IL23 + IL6. Total RNA was isolated and the gene expressions were measured by quantitative real-time PCR on the fifth day of the differentiation. RORC and TBX21 transcription factor expressions of cells derived from healthy donors [ (A) n = 10], rheumatoid arthritis (RA) [ (B) n = 10], and psoriatic arthritis (PsA) patients [ (C) n = 6] are shown in a logarithmic scale. Untreated naive cells served as controls. The median, minimum, and maximum values are indicated (Friedman test and pairwise Wilcoxon signed rank test * p
Figure Legend Snippet: RORC and TBX21 expression during T-helper 17 (Th17) differentiation. Naive CD4 + T cells were differentiated toward Th17-lane. Samples were treated with CD3/CD28/CAB (stim) or with stim + anti-IL4 antibody and the following four cytokine combinations to promote naive CD4 + T cell differentiation: transforming grow factor beta (TGF β ) + IL6, TGF β + IL6 + IL1 β , IL1 β + IL23, and IL1 β + IL23 + IL6. Total RNA was isolated and the gene expressions were measured by quantitative real-time PCR on the fifth day of the differentiation. RORC and TBX21 transcription factor expressions of cells derived from healthy donors [ (A) n = 10], rheumatoid arthritis (RA) [ (B) n = 10], and psoriatic arthritis (PsA) patients [ (C) n = 6] are shown in a logarithmic scale. Untreated naive cells served as controls. The median, minimum, and maximum values are indicated (Friedman test and pairwise Wilcoxon signed rank test * p

Techniques Used: Expressing, Cell Differentiation, Isolation, Real-time Polymerase Chain Reaction, Derivative Assay

40) Product Images from "Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit"

Article Title: Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit

Journal: eLife

doi: 10.7554/eLife.25517

CD11b+ cells isolated from mice with pathogenic CD4+ T cell transfer under stress condition have the potential of antigen presentation to CD4+ T cells without peptide addition. Co-culture supernatants of naïve 2D2 TCR transgenic T cells and CD11b+ cells isolated from SD+ MOG-T+ mice were collected and assessed using an ELISA specific for mouse IL-2. (n = 2 per group). Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. Statistical significance is denoted by asterisks (*p
Figure Legend Snippet: CD11b+ cells isolated from mice with pathogenic CD4+ T cell transfer under stress condition have the potential of antigen presentation to CD4+ T cells without peptide addition. Co-culture supernatants of naïve 2D2 TCR transgenic T cells and CD11b+ cells isolated from SD+ MOG-T+ mice were collected and assessed using an ELISA specific for mouse IL-2. (n = 2 per group). Mean scores ± SEM are shown. Statistical significance was determined by ANOVA tests. Statistical significance is denoted by asterisks (*p

Techniques Used: Isolation, Mouse Assay, Co-Culture Assay, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Related Articles

Cell Isolation:

Article Title: High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays
Article Snippet: .. Cells were obtained from a mouse spleen and two lymph nodes, which were ground through a filter, subjected to ACK lysis, purified with a CD8a+ T Cell Isolation Kit (Miltenyi), and then immediately seeded into wells and activated in RPMI 1640 with 10% FBS, 1X streptomycin/penicillin, and 55 μM β-mercaptoethanol (Gibco), using surface-bound anti-CD3 (Biolegend 145-2C11, coated at 5μg/mL) and soluble anti-CD28 (Biolegend, 37.51, 2 μg/mL) at 37°C under 5% CO2 for 22 h. After 22 h cells were transferred to new medium containing IL-2 (100 U/mL) and from there on passaged daily to a concentration of roughly 200,000 cells/mL, 2–3 hours prior to each measurement. .. Human blood buffy coat was obtained from Research Blood Components (Boston, MA) and PBMCs were isolated with Ficoll-Paque Plus (GE) using the manufacturer’s recommended protocol.

Synthesized:

Article Title: Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia
Article Snippet: .. Anti-human or mouse IgM (Jackson ImmunoResearch, West Grove, PA), CpG (Integrated DNA Technologies, Coralville, IA), anti-mouse CD3 (BioLegend), anti-mouse CD28 (BioLegend) and MTM (Enzo Life Sciences, Farmingdale, NY) are commercially sourced, while MTMox 32E was synthesized in house (Liu and Eckenrode et al, manuscript in preparation). .. Secreted cytokines were measured with ELISA MAX Standard Set Mouse or Human kits according to the manufacturer’s protocol (BioLegend).

Labeling:

Article Title: Arginase-1 is neither constitutively expressed in nor required for myeloid-derived suppressor cell (MDSC)-mediated inhibition of T cell proliferation
Article Snippet: .. Labeled splenocytes (6 × 105 ) or purified T cells (2 ×105 ) were plated in a 96-well plate pre-coated with anti-CD3 (1 μg/ml, clone 145-2C11, Biolegend) in the presence of soluble anti-CD28 (0.5 μg/ml, clone 37.51, Biolegend) in RPMI 1640 with 10% FBS for 4 days (37°C, 5% CO2 ). .. For Concanavalin A (ConA), PMA/Ionomycin, or IL-2-induced T cell proliferation, CFSE labeled splenocytes or purified T cells were treated with 1 μg/mL ConA, 20 ng/ml PMA/60 ng/ml ionomycin or 1000 IU/ml IL-2, respectively, in RPMI 1640 with 10 % FBS for 4 days (37°C, 5% CO2).

Purification:

Article Title: Arginase-1 is neither constitutively expressed in nor required for myeloid-derived suppressor cell (MDSC)-mediated inhibition of T cell proliferation
Article Snippet: .. Labeled splenocytes (6 × 105 ) or purified T cells (2 ×105 ) were plated in a 96-well plate pre-coated with anti-CD3 (1 μg/ml, clone 145-2C11, Biolegend) in the presence of soluble anti-CD28 (0.5 μg/ml, clone 37.51, Biolegend) in RPMI 1640 with 10% FBS for 4 days (37°C, 5% CO2 ). .. For Concanavalin A (ConA), PMA/Ionomycin, or IL-2-induced T cell proliferation, CFSE labeled splenocytes or purified T cells were treated with 1 μg/mL ConA, 20 ng/ml PMA/60 ng/ml ionomycin or 1000 IU/ml IL-2, respectively, in RPMI 1640 with 10 % FBS for 4 days (37°C, 5% CO2).

Article Title: High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays
Article Snippet: .. Cells were obtained from a mouse spleen and two lymph nodes, which were ground through a filter, subjected to ACK lysis, purified with a CD8a+ T Cell Isolation Kit (Miltenyi), and then immediately seeded into wells and activated in RPMI 1640 with 10% FBS, 1X streptomycin/penicillin, and 55 μM β-mercaptoethanol (Gibco), using surface-bound anti-CD3 (Biolegend 145-2C11, coated at 5μg/mL) and soluble anti-CD28 (Biolegend, 37.51, 2 μg/mL) at 37°C under 5% CO2 for 22 h. After 22 h cells were transferred to new medium containing IL-2 (100 U/mL) and from there on passaged daily to a concentration of roughly 200,000 cells/mL, 2–3 hours prior to each measurement. .. Human blood buffy coat was obtained from Research Blood Components (Boston, MA) and PBMCs were isolated with Ficoll-Paque Plus (GE) using the manufacturer’s recommended protocol.

Concentration Assay:

Article Title: High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays
Article Snippet: .. Cells were obtained from a mouse spleen and two lymph nodes, which were ground through a filter, subjected to ACK lysis, purified with a CD8a+ T Cell Isolation Kit (Miltenyi), and then immediately seeded into wells and activated in RPMI 1640 with 10% FBS, 1X streptomycin/penicillin, and 55 μM β-mercaptoethanol (Gibco), using surface-bound anti-CD3 (Biolegend 145-2C11, coated at 5μg/mL) and soluble anti-CD28 (Biolegend, 37.51, 2 μg/mL) at 37°C under 5% CO2 for 22 h. After 22 h cells were transferred to new medium containing IL-2 (100 U/mL) and from there on passaged daily to a concentration of roughly 200,000 cells/mL, 2–3 hours prior to each measurement. .. Human blood buffy coat was obtained from Research Blood Components (Boston, MA) and PBMCs were isolated with Ficoll-Paque Plus (GE) using the manufacturer’s recommended protocol.

Lysis:

Article Title: High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays
Article Snippet: .. Cells were obtained from a mouse spleen and two lymph nodes, which were ground through a filter, subjected to ACK lysis, purified with a CD8a+ T Cell Isolation Kit (Miltenyi), and then immediately seeded into wells and activated in RPMI 1640 with 10% FBS, 1X streptomycin/penicillin, and 55 μM β-mercaptoethanol (Gibco), using surface-bound anti-CD3 (Biolegend 145-2C11, coated at 5μg/mL) and soluble anti-CD28 (Biolegend, 37.51, 2 μg/mL) at 37°C under 5% CO2 for 22 h. After 22 h cells were transferred to new medium containing IL-2 (100 U/mL) and from there on passaged daily to a concentration of roughly 200,000 cells/mL, 2–3 hours prior to each measurement. .. Human blood buffy coat was obtained from Research Blood Components (Boston, MA) and PBMCs were isolated with Ficoll-Paque Plus (GE) using the manufacturer’s recommended protocol.

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  • 93
    BioLegend icam 1
    NPEC 470 and UPEC CFT073 enhance <t>ICAM-1</t> expression in the epididymis during acute stages of infection. Epididymal sections immunolabeled for detection of ICAM-1/CD54. Epididymides were harvested from C57BL/6N mice at day 3 (A to C) or day 7 (D to F) postinjection
    Icam 1, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    BioLegend allophycocyanin conjugated goat anti mouse igg
    Generation of B16F10 clones containing TS-MAC or MAC4 . ( a ) PCR analysis with the primers for B16F10 clones containing TS-MAC or MAC4 from microcell-mediated chromosome transfer (MMCT). ( b ) A representative image of FISH analysis of B16F10/TS-MAC#1. Blue indicates 4′,6-diamidino-2-phenylindole signals. The rhodamine (red) signal indicates the centromere sequence of mouse chromosomes. The fluorescein isothiocyanate signal (green), which was observed as a yellow dot, indicates the inserted PAC vector. The yellow arrow shows TS-MAC. An elongated TS-MAC is shown in the inset. ( c ) Flow cytometric analysis of B16F10 clones containing TS-MAC or MAC4 using antibodies against MHC H2-K(d) or -K(b). The gray histogram shows the counts of MHC H2-K(b) polyclonal mouse anti-goat <t>IgG</t> conjugated with <t>allophycocyanin</t> (APC) as a negative control. Red histograms show the counts of MHC H2-K(d) detected by a polyclonal mouse anti-goat IgG conjugated with APC.
    Allophycocyanin Conjugated Goat Anti Mouse Igg, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    BioLegend apc conjugated goat anti mouse igg2a antibody
    BDLF3 induces more rapid internalization and a delayed appearance of both MHC class I and class II molecules at the cell surface. Internalization and appearance assays were performed on MJS cells transiently expressing control-GFP or BDLF3-GFP. The GFP + population was used to gate BDLF3-expressing cells. Internalization and appearance assays were performed on cells pretreated on ice with saturating amounts of anti-MHC class I antibody or anti-MHC class II antibody. Cells were then washed and incubated at 37°C for up to 60 min. (A) For the internalization assay, viable cells harvested at each time point were stained with an <t>APC-conjugated</t> goat anti-mouse <t>IgG</t> antibody and analyzed using flow cytometry at the indicated times; this identified the prelabeled antibody-bound MHC molecules that remained at the surface, while endocytosed labeled MHC molecules were not detected on the viable cells. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. (B) For the appearance assays, newly arrived MHC-I and MHC-II molecules, which were not prelabeled with unconjugated antibodies, were detected by staining with an APC-conjugated anti-MHC class I antibody or anti-MHC class II antibody. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. Results are representative of three independent experiments.
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    92
    BioLegend cd3e igg complexes
    TCR sensitization via Scn5a expression correlates with more rapid and robust TCR-proximal signaling, using PLCγ phosphorylation and Ca2+ flux as readouts (A) Phosphorylation of PLCγ (relative to b-actin) in response to sub-maximal CD3 crosslinking (10µg/mL) was measured at 30 sec., 1 min., 3 min., 5 min., 10 min. and 15 min., in 1.5×10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. Fold change (a ratio of Scn5a+CD4-Cre+ to Scn5a+CD4-Cre- pPLCγ:b-actin maximum levels) is quantified on the right. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts. (B) Ca2+ flux was measured in 10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. <t>CD3e/IgG</t> complexes were added at 5 minutes, except in the control wells, in which Ca2+ buffer alone was used. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts.
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    NPEC 470 and UPEC CFT073 enhance ICAM-1 expression in the epididymis during acute stages of infection. Epididymal sections immunolabeled for detection of ICAM-1/CD54. Epididymides were harvested from C57BL/6N mice at day 3 (A to C) or day 7 (D to F) postinjection

    Journal: Infection and Immunity

    Article Title: Uropathogenic Escherichia coli Modulates Innate Immunity To Suppress Th1-Mediated Inflammatory Responses during Infectious Epididymitis

    doi: 10.1128/IAI.01373-13

    Figure Lengend Snippet: NPEC 470 and UPEC CFT073 enhance ICAM-1 expression in the epididymis during acute stages of infection. Epididymal sections immunolabeled for detection of ICAM-1/CD54. Epididymides were harvested from C57BL/6N mice at day 3 (A to C) or day 7 (D to F) postinjection

    Article Snippet: Secondary antibodies for detection of CD3 and ICAM-1 (Dylight 488 goat anti-rat IgG [Biolegend], dilution 1:800) and F4/80 (fluorescein isothiocyanate [FITC]-conjugated donkey anti-rabbit IgG [Merck Millipore, Darmstadt, Germany], dilution 1:1,000) were incubated for 1 h, respectively.

    Techniques: Expressing, Infection, Immunolabeling, Mouse Assay

    Generation of B16F10 clones containing TS-MAC or MAC4 . ( a ) PCR analysis with the primers for B16F10 clones containing TS-MAC or MAC4 from microcell-mediated chromosome transfer (MMCT). ( b ) A representative image of FISH analysis of B16F10/TS-MAC#1. Blue indicates 4′,6-diamidino-2-phenylindole signals. The rhodamine (red) signal indicates the centromere sequence of mouse chromosomes. The fluorescein isothiocyanate signal (green), which was observed as a yellow dot, indicates the inserted PAC vector. The yellow arrow shows TS-MAC. An elongated TS-MAC is shown in the inset. ( c ) Flow cytometric analysis of B16F10 clones containing TS-MAC or MAC4 using antibodies against MHC H2-K(d) or -K(b). The gray histogram shows the counts of MHC H2-K(b) polyclonal mouse anti-goat IgG conjugated with allophycocyanin (APC) as a negative control. Red histograms show the counts of MHC H2-K(d) detected by a polyclonal mouse anti-goat IgG conjugated with APC.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy

    doi: 10.1038/mtna.2015.45

    Figure Lengend Snippet: Generation of B16F10 clones containing TS-MAC or MAC4 . ( a ) PCR analysis with the primers for B16F10 clones containing TS-MAC or MAC4 from microcell-mediated chromosome transfer (MMCT). ( b ) A representative image of FISH analysis of B16F10/TS-MAC#1. Blue indicates 4′,6-diamidino-2-phenylindole signals. The rhodamine (red) signal indicates the centromere sequence of mouse chromosomes. The fluorescein isothiocyanate signal (green), which was observed as a yellow dot, indicates the inserted PAC vector. The yellow arrow shows TS-MAC. An elongated TS-MAC is shown in the inset. ( c ) Flow cytometric analysis of B16F10 clones containing TS-MAC or MAC4 using antibodies against MHC H2-K(d) or -K(b). The gray histogram shows the counts of MHC H2-K(b) polyclonal mouse anti-goat IgG conjugated with allophycocyanin (APC) as a negative control. Red histograms show the counts of MHC H2-K(d) detected by a polyclonal mouse anti-goat IgG conjugated with APC.

    Article Snippet: The cells were washed with PBS, resuspended in 100 µl PBS, and incubated with allophycocyanin-conjugated goat anti-mouse IgG (Poly4053; Biolegend) for 1 hour at 4 °C.

    Techniques: Clone Assay, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Sequencing, Plasmid Preparation, Flow Cytometry, Negative Control

    BDLF3 induces more rapid internalization and a delayed appearance of both MHC class I and class II molecules at the cell surface. Internalization and appearance assays were performed on MJS cells transiently expressing control-GFP or BDLF3-GFP. The GFP + population was used to gate BDLF3-expressing cells. Internalization and appearance assays were performed on cells pretreated on ice with saturating amounts of anti-MHC class I antibody or anti-MHC class II antibody. Cells were then washed and incubated at 37°C for up to 60 min. (A) For the internalization assay, viable cells harvested at each time point were stained with an APC-conjugated goat anti-mouse IgG antibody and analyzed using flow cytometry at the indicated times; this identified the prelabeled antibody-bound MHC molecules that remained at the surface, while endocytosed labeled MHC molecules were not detected on the viable cells. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. (B) For the appearance assays, newly arrived MHC-I and MHC-II molecules, which were not prelabeled with unconjugated antibodies, were detected by staining with an APC-conjugated anti-MHC class I antibody or anti-MHC class II antibody. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. Results are representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

    doi: 10.1128/JVI.02183-15

    Figure Lengend Snippet: BDLF3 induces more rapid internalization and a delayed appearance of both MHC class I and class II molecules at the cell surface. Internalization and appearance assays were performed on MJS cells transiently expressing control-GFP or BDLF3-GFP. The GFP + population was used to gate BDLF3-expressing cells. Internalization and appearance assays were performed on cells pretreated on ice with saturating amounts of anti-MHC class I antibody or anti-MHC class II antibody. Cells were then washed and incubated at 37°C for up to 60 min. (A) For the internalization assay, viable cells harvested at each time point were stained with an APC-conjugated goat anti-mouse IgG antibody and analyzed using flow cytometry at the indicated times; this identified the prelabeled antibody-bound MHC molecules that remained at the surface, while endocytosed labeled MHC molecules were not detected on the viable cells. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. (B) For the appearance assays, newly arrived MHC-I and MHC-II molecules, which were not prelabeled with unconjugated antibodies, were detected by staining with an APC-conjugated anti-MHC class I antibody or anti-MHC class II antibody. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. Results are representative of three independent experiments.

    Article Snippet: The level of W6/32 or L243 MAb remaining at the cell surface was then analyzed by staining cells with APC-conjugated goat anti-mouse IgG2a antibody (Biolegend).

    Techniques: Expressing, Incubation, Staining, Flow Cytometry, Cytometry, Labeling, Fluorescence

    TCR sensitization via Scn5a expression correlates with more rapid and robust TCR-proximal signaling, using PLCγ phosphorylation and Ca2+ flux as readouts (A) Phosphorylation of PLCγ (relative to b-actin) in response to sub-maximal CD3 crosslinking (10µg/mL) was measured at 30 sec., 1 min., 3 min., 5 min., 10 min. and 15 min., in 1.5×10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. Fold change (a ratio of Scn5a+CD4-Cre+ to Scn5a+CD4-Cre- pPLCγ:b-actin maximum levels) is quantified on the right. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts. (B) Ca2+ flux was measured in 10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. CD3e/IgG complexes were added at 5 minutes, except in the control wells, in which Ca2+ buffer alone was used. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Tuning T Cell Signaling Sensitivity Alters the Behavior of CD4+ T Cells During an Immune Response

    doi: 10.4049/jimmunol.1701422

    Figure Lengend Snippet: TCR sensitization via Scn5a expression correlates with more rapid and robust TCR-proximal signaling, using PLCγ phosphorylation and Ca2+ flux as readouts (A) Phosphorylation of PLCγ (relative to b-actin) in response to sub-maximal CD3 crosslinking (10µg/mL) was measured at 30 sec., 1 min., 3 min., 5 min., 10 min. and 15 min., in 1.5×10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. Fold change (a ratio of Scn5a+CD4-Cre+ to Scn5a+CD4-Cre- pPLCγ:b-actin maximum levels) is quantified on the right. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts. (B) Ca2+ flux was measured in 10 6 CD4+ enriched T naïve splenocytes from both Scn5a+CD4-Cre+ mice and their Scn5a+CD4-Cre- littermates. CD3e/IgG complexes were added at 5 minutes, except in the control wells, in which Ca2+ buffer alone was used. Data are representative of three separate experiments, with splenocytes from two mice pooled within cohorts.

    Article Snippet: CD3e/IgG complexes (CD3ε, clone 2C11, Biolegend; Biotin Goat anti-hamster, Biolegend) were added at the 5-minute time point.

    Techniques: Expressing, Size-exclusion Chromatography, Mouse Assay