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Becton Dickinson facscalibur flow cytometer
Vanillic acid inhibits the proliferation of HCT116 cells via blocking cell cycle progression in the G1 phase. ( A ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Subsequently, cell cycle status was analyzed by by <t>FACSCalibur</t> flow cytometry. ( B ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Cyclin D1 and c-Myc were detected by Western blot analysis. Levels of tubulin were used as a loading control. ( C ) Data were shown as mean ± SD ( n = 3). ** p
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1) Product Images from "Vanillic Acid Suppresses HIF-1α Expression via Inhibition of mTOR/p70S6K/4E-BP1 and Raf/MEK/ERK Pathways in Human Colon Cancer HCT116 Cells"

Article Title: Vanillic Acid Suppresses HIF-1α Expression via Inhibition of mTOR/p70S6K/4E-BP1 and Raf/MEK/ERK Pathways in Human Colon Cancer HCT116 Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20030465

Vanillic acid inhibits the proliferation of HCT116 cells via blocking cell cycle progression in the G1 phase. ( A ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Subsequently, cell cycle status was analyzed by by FACSCalibur flow cytometry. ( B ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Cyclin D1 and c-Myc were detected by Western blot analysis. Levels of tubulin were used as a loading control. ( C ) Data were shown as mean ± SD ( n = 3). ** p
Figure Legend Snippet: Vanillic acid inhibits the proliferation of HCT116 cells via blocking cell cycle progression in the G1 phase. ( A ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Subsequently, cell cycle status was analyzed by by FACSCalibur flow cytometry. ( B ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Cyclin D1 and c-Myc were detected by Western blot analysis. Levels of tubulin were used as a loading control. ( C ) Data were shown as mean ± SD ( n = 3). ** p

Techniques Used: Blocking Assay, Concentration Assay, Cell Culture, Flow Cytometry, Cytometry, Western Blot

2) Product Images from "Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection"

Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection

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

doi:

CD8 T cells responding to lytic (ORF65 131–140 /D d ) or latent (M2 91–99 /K d ) Ags have different abilities to kill peptide-loaded targets in vivo. Latently infected BALB/c mice ( > 40 days postinfection with MHV-68) were given 2 × 10 7 fluorescently labeled splenocytes i.v. as described in Materials and Methods . In the first experiment ( A ) killing was analyzed 16 or 40 h later, whereas in the second experiment ( B ) killing was analyzed 40 or 64 h postinjection. To measure Ag-specific killing, splenocytes were harvested, incubated with 20 µg/ml 7-AAD for 15 min and then analyzed using a FACSCalibur cytometer. Specific lysis was then determined as described in Materials and Methods and the killing of M2 91–99 (○) and ORF65 131–140 (△) labeled cells is shown in the graph. Each symbol represents one mouse with the bar indicating the average for that group. p values for a given time-point are and were determined using Student’s t test.
Figure Legend Snippet: CD8 T cells responding to lytic (ORF65 131–140 /D d ) or latent (M2 91–99 /K d ) Ags have different abilities to kill peptide-loaded targets in vivo. Latently infected BALB/c mice ( > 40 days postinfection with MHV-68) were given 2 × 10 7 fluorescently labeled splenocytes i.v. as described in Materials and Methods . In the first experiment ( A ) killing was analyzed 16 or 40 h later, whereas in the second experiment ( B ) killing was analyzed 40 or 64 h postinjection. To measure Ag-specific killing, splenocytes were harvested, incubated with 20 µg/ml 7-AAD for 15 min and then analyzed using a FACSCalibur cytometer. Specific lysis was then determined as described in Materials and Methods and the killing of M2 91–99 (○) and ORF65 131–140 (△) labeled cells is shown in the graph. Each symbol represents one mouse with the bar indicating the average for that group. p values for a given time-point are and were determined using Student’s t test.

Techniques Used: In Vivo, Infection, Mouse Assay, Labeling, Incubation, Cytometry, Lysis

Survival of lytic or latent Ag-specific CD8 T cells in response to IL-7 or IL-15 in vitro. Spleen cells from BALB/c mice infected for > 40 days with MHV-68 were labeled with CFSE then placed in culture with 25 ng/ml recombinant mouse IL-15 or 25 ng/ml recombinant mouse IL-7. Four days later, the cells were harvested, counted, and stained with tetramers consisting of the M2 91–99 /K d epitope (○), or the ORF65 131–140 /D d epitope (●) and anti-CD8a Ab. To identify dead cells, samples were incubated with 20 µg/ml 7-AAD for 15 min before analyzing on a FACSCalibur cytometer. A , Gating used for analysis of the data. B , The number of live tetramer-positive cells in the different treatment groups relative to the numbers obtained after culture with medium alone. Each point represents data from a different experiment, a total of six experiments were performed. Values for p were calculated using Student’s t test.
Figure Legend Snippet: Survival of lytic or latent Ag-specific CD8 T cells in response to IL-7 or IL-15 in vitro. Spleen cells from BALB/c mice infected for > 40 days with MHV-68 were labeled with CFSE then placed in culture with 25 ng/ml recombinant mouse IL-15 or 25 ng/ml recombinant mouse IL-7. Four days later, the cells were harvested, counted, and stained with tetramers consisting of the M2 91–99 /K d epitope (○), or the ORF65 131–140 /D d epitope (●) and anti-CD8a Ab. To identify dead cells, samples were incubated with 20 µg/ml 7-AAD for 15 min before analyzing on a FACSCalibur cytometer. A , Gating used for analysis of the data. B , The number of live tetramer-positive cells in the different treatment groups relative to the numbers obtained after culture with medium alone. Each point represents data from a different experiment, a total of six experiments were performed. Values for p were calculated using Student’s t test.

Techniques Used: In Vitro, Mouse Assay, Infection, Labeling, Recombinant, Staining, Incubation, Cytometry

3) Product Images from "Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells"

Article Title: Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01405

Comparison of PTA and ZOL in the induction of IFN-γ from Vγ2Vδ2 T cells. (A) Determination of IFN-γ produced by Vγ2Vδ2 T cells in response to lung cancer cell lines pulsed with PTA or ZOL. Human lung cancer cells, PC-9, PC-6, H1975, and H520 (4 × 10 5 cells), pretreated with PTA (•) at concentrations of 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 nM, or ZOL (⚬) at concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 μM were challenged by PTA-expanded Vγ2Vδ2 T cells (4 × 10 5 cells). After incubation for 16 h, the culture supernatants were examined for IFN-γ levels through ELISA. (B) Intracellular staining of IFN-γ in Vγ2Vδ2 T cells in response to human lung cancer cells pretreated with PTA or ZOL. PC-9 human lung cancer cells (1 × 10 6 cells/ml) were treated with 1 ml of the complete RPMI1640 medium or the medium containing 1 μM PTA or with 1 mM ZOL at 37°C with 5% CO 2 for 2 h. Then, the cells were examined for intracellular IFN-γ using a FACSCalibur flow cytometer and the cell population was visualized using FlowJo ver. 10.
Figure Legend Snippet: Comparison of PTA and ZOL in the induction of IFN-γ from Vγ2Vδ2 T cells. (A) Determination of IFN-γ produced by Vγ2Vδ2 T cells in response to lung cancer cell lines pulsed with PTA or ZOL. Human lung cancer cells, PC-9, PC-6, H1975, and H520 (4 × 10 5 cells), pretreated with PTA (•) at concentrations of 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 nM, or ZOL (⚬) at concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 μM were challenged by PTA-expanded Vγ2Vδ2 T cells (4 × 10 5 cells). After incubation for 16 h, the culture supernatants were examined for IFN-γ levels through ELISA. (B) Intracellular staining of IFN-γ in Vγ2Vδ2 T cells in response to human lung cancer cells pretreated with PTA or ZOL. PC-9 human lung cancer cells (1 × 10 6 cells/ml) were treated with 1 ml of the complete RPMI1640 medium or the medium containing 1 μM PTA or with 1 mM ZOL at 37°C with 5% CO 2 for 2 h. Then, the cells were examined for intracellular IFN-γ using a FACSCalibur flow cytometer and the cell population was visualized using FlowJo ver. 10.

Techniques Used: Produced, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

4) Product Images from "Heparan sulfate structure is influenced by the ER-Golgi dynamics of its modifying enzymes"

Article Title: Heparan sulfate structure is influenced by the ER-Golgi dynamics of its modifying enzymes

Journal: bioRxiv

doi: 10.1101/2020.01.23.916940

Protein and gene expression of components of HS biosynthesis in presence of heparin. (A) Protein expression of HS-modifying enzymes (NDST1, C5-epimerase, HS2ST and HS3ST5) in transfected cells previously treated with heparin was evaluated by flow cytometry using antibodies specific for each enzyme. Following incubation with primary antibodies, cells were incubated with secondary antibody conjugated with Alexa Fluor® 633 and analyzed on FACSCalibur flow cytometer. (B) PAPS synthases mRNA level in EC-HS3ST5 cells treated with heparin was analyzed by real-time. The results were expressed as mean ± standard deviation. (Right panel) Heat map was generated of mean values obtained in the gene expression assays. High and low expression are shown in red and blue colors. *, Differences statistically significant, P
Figure Legend Snippet: Protein and gene expression of components of HS biosynthesis in presence of heparin. (A) Protein expression of HS-modifying enzymes (NDST1, C5-epimerase, HS2ST and HS3ST5) in transfected cells previously treated with heparin was evaluated by flow cytometry using antibodies specific for each enzyme. Following incubation with primary antibodies, cells were incubated with secondary antibody conjugated with Alexa Fluor® 633 and analyzed on FACSCalibur flow cytometer. (B) PAPS synthases mRNA level in EC-HS3ST5 cells treated with heparin was analyzed by real-time. The results were expressed as mean ± standard deviation. (Right panel) Heat map was generated of mean values obtained in the gene expression assays. High and low expression are shown in red and blue colors. *, Differences statistically significant, P

Techniques Used: Expressing, Transfection, Flow Cytometry, Incubation, Papanicolaou Stain, Standard Deviation, Generated

5) Product Images from "Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms"

Article Title: Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.695

Gemcitabine blocks reovirus-induced early recruitment of cancer-associated MDSCs in cancer environment through means other than direct oncolysis. ( A ) Female C57BL/6 mice were implanted i.p. with ID8 cells and then monitored for the development of PC. Then, these mice were injected with regimen of PBS/GEM/Reo alone or in combination as shown in the schematic. ( B ) The ascites were harvested and stained to detect the percentage of MDSCs (Gr-1+/CD11b+ cells) by flow cytometry at the respective time points and as shown in a representative example. The events were collected with same settings of the acquisition mode in FACSCalibur, demonstrating the gradient of CD11b expression between various samples; however, the statistical data include all relevant events in the analysis. The cumulative data from both ascites and spleen shown in ( C ) represent the fold increase/decrease in the percentages of MDSCs after normalising against PBS alone-treated control at the respective day post first injection (d.p.f.i.) or day post last injection (d.p.l.i.). The data are representative of n =3 except for Reo+GEM at 3 d.p.l.i., which is n =1. MDSCs were isolated from the ascites of OC-bearing mice and cultured in 50% cell-free ascites fluid and complete RPMI-1640 ( D ), while those isolated from the bone marrow of naive mice were cultured in complete RPMI-1640 ( E ). Isolated MDSCs were then treated with Reo (1 MOI) and GEM (1 μ M ) alone or in combination as indicated. Cells were harvested at 24 h and stained with GR-1+/CD11b+ antibodies along with apoptosis marker (Annexin-V) followed by flow cytometry. Data are representative of three independent experiments. Asterisks shown immediately on top of the bars represent the P- values obtained by comparing the respective Reo+PBS, GEM+PBS, and Reo+GEM-treated groups against PBS control. Statistical analysis was performed with two-tailed, Student's t -test with 95% CI; ns= P > 0.05; * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001. Error bars are defined as mean+s.d.
Figure Legend Snippet: Gemcitabine blocks reovirus-induced early recruitment of cancer-associated MDSCs in cancer environment through means other than direct oncolysis. ( A ) Female C57BL/6 mice were implanted i.p. with ID8 cells and then monitored for the development of PC. Then, these mice were injected with regimen of PBS/GEM/Reo alone or in combination as shown in the schematic. ( B ) The ascites were harvested and stained to detect the percentage of MDSCs (Gr-1+/CD11b+ cells) by flow cytometry at the respective time points and as shown in a representative example. The events were collected with same settings of the acquisition mode in FACSCalibur, demonstrating the gradient of CD11b expression between various samples; however, the statistical data include all relevant events in the analysis. The cumulative data from both ascites and spleen shown in ( C ) represent the fold increase/decrease in the percentages of MDSCs after normalising against PBS alone-treated control at the respective day post first injection (d.p.f.i.) or day post last injection (d.p.l.i.). The data are representative of n =3 except for Reo+GEM at 3 d.p.l.i., which is n =1. MDSCs were isolated from the ascites of OC-bearing mice and cultured in 50% cell-free ascites fluid and complete RPMI-1640 ( D ), while those isolated from the bone marrow of naive mice were cultured in complete RPMI-1640 ( E ). Isolated MDSCs were then treated with Reo (1 MOI) and GEM (1 μ M ) alone or in combination as indicated. Cells were harvested at 24 h and stained with GR-1+/CD11b+ antibodies along with apoptosis marker (Annexin-V) followed by flow cytometry. Data are representative of three independent experiments. Asterisks shown immediately on top of the bars represent the P- values obtained by comparing the respective Reo+PBS, GEM+PBS, and Reo+GEM-treated groups against PBS control. Statistical analysis was performed with two-tailed, Student's t -test with 95% CI; ns= P > 0.05; * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001. Error bars are defined as mean+s.d.

Techniques Used: Mouse Assay, Injection, Staining, Flow Cytometry, Cytometry, Expressing, Isolation, Cell Culture, Marker, Two Tailed Test

6) Product Images from "Major histocompatibility complex class I molecules with super-enhanced CD8 binding properties bypass the requirement for cognate TCR recognition and non-specifically activate cytotoxic T lymphocytes 1"

Article Title: Major histocompatibility complex class I molecules with super-enhanced CD8 binding properties bypass the requirement for cognate TCR recognition and non-specifically activate cytotoxic T lymphocytes 1

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

doi: 10.4049/jimmunol.0902398

Non-cognate A2/K b -mediated CTL activation and tetramer binding is not influenced by MHCI restriction A. 2.5×10 5 PBMC were suspended in 250μl FACS buffer (2% FCS/PBS) and stained with FITC-conjugated anti-A2 and 7-AAD for 30 minutes on ice, then washed twice and resuspended in PBS. For pMHCI tetramer staining experiments, 2.5×10 5 PBMC were suspended in 50μl FACS buffer (2% FCS/PBS) and incubated +/− 10μg/ml unconjugated anti-CD8 for 20 minutes on ice, then stained with 10μg/ml of the PE-conjugated tetramers A2 ILAKFLHWL or A2/K b ILAKFLHWL for 45 minutes on ice. After washing, cells were subsequently stained with APC-conjugated anti-CD8 and 7-AAD, washed again and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software. B. 2.5×10 4 CTL were incubated for 12 hours at 37°C with 10 5 unpulsed C1R cells expressing either A2 or A2/K b on the cell surface. The following CTL clones were used: (i) the HLA A*6801-restricted CTL clone c23, specific for the HIV-1 Tat-derived epitope ITKGLGISYGR (residues 38-48); (ii) the HLA B*0702-restricted CTL clone KD4, specific for the EBV EBNA3A-derived epitope RPPIFIRRL (residues 379-387); (iii) the HLA B*0801-restricted CTL clone LC13, specific for the EBV EBNA3A-derived epitope FLRGRAYGL (residues 339-347); and, (iv) the HLA B*3508-restricted CTL clone SB27, specific for the EBV BZLF1-derived epitope LPEPLPQGQLTAY (residues 52-64). Supernatant was subsequently assayed for MIP-1β content by ELISA. The mean ± SD of two replicate assays is shown.
Figure Legend Snippet: Non-cognate A2/K b -mediated CTL activation and tetramer binding is not influenced by MHCI restriction A. 2.5×10 5 PBMC were suspended in 250μl FACS buffer (2% FCS/PBS) and stained with FITC-conjugated anti-A2 and 7-AAD for 30 minutes on ice, then washed twice and resuspended in PBS. For pMHCI tetramer staining experiments, 2.5×10 5 PBMC were suspended in 50μl FACS buffer (2% FCS/PBS) and incubated +/− 10μg/ml unconjugated anti-CD8 for 20 minutes on ice, then stained with 10μg/ml of the PE-conjugated tetramers A2 ILAKFLHWL or A2/K b ILAKFLHWL for 45 minutes on ice. After washing, cells were subsequently stained with APC-conjugated anti-CD8 and 7-AAD, washed again and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software. B. 2.5×10 4 CTL were incubated for 12 hours at 37°C with 10 5 unpulsed C1R cells expressing either A2 or A2/K b on the cell surface. The following CTL clones were used: (i) the HLA A*6801-restricted CTL clone c23, specific for the HIV-1 Tat-derived epitope ITKGLGISYGR (residues 38-48); (ii) the HLA B*0702-restricted CTL clone KD4, specific for the EBV EBNA3A-derived epitope RPPIFIRRL (residues 379-387); (iii) the HLA B*0801-restricted CTL clone LC13, specific for the EBV EBNA3A-derived epitope FLRGRAYGL (residues 339-347); and, (iv) the HLA B*3508-restricted CTL clone SB27, specific for the EBV BZLF1-derived epitope LPEPLPQGQLTAY (residues 52-64). Supernatant was subsequently assayed for MIP-1β content by ELISA. The mean ± SD of two replicate assays is shown.

Techniques Used: CTL Assay, Activation Assay, Binding Assay, FACS, Staining, Incubation, Flow Cytometry, Cytometry, Software, Expressing, Clone Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay

A2/K b tetramers bind the majority of CTL in peripheral blood A. 2.5×10 5 PBMC from an A2 + donor were stained with PerCP-conjugated anti-CD8, 7-AAD and either FITC-conjugated anti-αβ-TCR, APC-conjugated anti-CD56 or PE-conjugated anti-γδ-TCR for 30 minutes on ice, washed twice and resuspended in PBS. B. 2.5×10 5 A2 + PBMC were stained with 10μg/ml of the PE-conjugated tetramers A2 ILAKFLHWL or A2/K b ILAKFLHWL for 20 minutes at 37°C. After washing, cells were subsequently stained with 7-AAD and either FITC-conjugated anti-γδ-TCR or FITC-conjugated anti-CD56 for 30 minutes on ice, washed twice and resuspended in PBS. C. 2.5×10 5 A2 + PBMC were stained with 10μg/ml of the PE-conjugated tetramers A2 ILAKFLHWL or A2/K b ILAKFLHWL for 20 minutes at 37°C. After washing, cells were stained with APC-conjugated anti-CD8, FITC-conjugated anti-αβ-TCR and 7-AAD for 30 minutes on ice, washed twice and resuspended in PBS. In A , B and C , data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.
Figure Legend Snippet: A2/K b tetramers bind the majority of CTL in peripheral blood A. 2.5×10 5 PBMC from an A2 + donor were stained with PerCP-conjugated anti-CD8, 7-AAD and either FITC-conjugated anti-αβ-TCR, APC-conjugated anti-CD56 or PE-conjugated anti-γδ-TCR for 30 minutes on ice, washed twice and resuspended in PBS. B. 2.5×10 5 A2 + PBMC were stained with 10μg/ml of the PE-conjugated tetramers A2 ILAKFLHWL or A2/K b ILAKFLHWL for 20 minutes at 37°C. After washing, cells were subsequently stained with 7-AAD and either FITC-conjugated anti-γδ-TCR or FITC-conjugated anti-CD56 for 30 minutes on ice, washed twice and resuspended in PBS. C. 2.5×10 5 A2 + PBMC were stained with 10μg/ml of the PE-conjugated tetramers A2 ILAKFLHWL or A2/K b ILAKFLHWL for 20 minutes at 37°C. After washing, cells were stained with APC-conjugated anti-CD8, FITC-conjugated anti-αβ-TCR and 7-AAD for 30 minutes on ice, washed twice and resuspended in PBS. In A , B and C , data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.

Techniques Used: CTL Assay, Staining, Flow Cytometry, Cytometry, Software

Non-specific A2/K b tetramer binding is influenced by CD8 cell surface density A and B. 2×10 5 293T cells were incubated +/− 10 μg/ml of the PE-conjugated tetramers A2 D227K/T228A ILAKFLHWL, A2 ILAKFLHWL or A2/K b ILAKFLHWL for 20 minutes at 37°C, then stained with 7-AAD and either FITC-conjugated anti-CD8 or PE-conjugated anti-CD8β for 30 minutes on ice, washed twice and resuspended in PBS. C. 2.5×10 5 PBMC were stained with PerCP-conjugated anti-CD8, 7-AAD and either FITC-conjugated anti-αβ-TCR, APC-conjugated anti-CD56 or PE-conjugated anti-γδ-TCR for 30 minutes on ice, washed twice and resuspended in PBS. In A , B and C , data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.
Figure Legend Snippet: Non-specific A2/K b tetramer binding is influenced by CD8 cell surface density A and B. 2×10 5 293T cells were incubated +/− 10 μg/ml of the PE-conjugated tetramers A2 D227K/T228A ILAKFLHWL, A2 ILAKFLHWL or A2/K b ILAKFLHWL for 20 minutes at 37°C, then stained with 7-AAD and either FITC-conjugated anti-CD8 or PE-conjugated anti-CD8β for 30 minutes on ice, washed twice and resuspended in PBS. C. 2.5×10 5 PBMC were stained with PerCP-conjugated anti-CD8, 7-AAD and either FITC-conjugated anti-αβ-TCR, APC-conjugated anti-CD56 or PE-conjugated anti-γδ-TCR for 30 minutes on ice, washed twice and resuspended in PBS. In A , B and C , data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.

Techniques Used: Binding Assay, Incubation, Staining, Flow Cytometry, Cytometry, Software

A2/K b tetramers can activate CTL in the absence of a specific TCR/pMHCI interaction A. 10 5 003 CTL were suspended in 20μl PBS and stained with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV at the indicated concentrations and 7-AAD for 20 minutes at 37°C. Cells were then washed twice and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software. B. 10 5 003 CTL were suspended in 40μl R2 with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV at the indicated concentrations for 30 minutes at 37°C. Cells were subsequently stained with FITC-conjugated anti-αβ-TCR, 7-AAD and APC-conjugated anti-CD8 for 30 minutes on ice in azide buffer (0.1% azide/2% FCS/PBS). After two washes, data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software. C. 5×10 5 003 CTL were incubated with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV at the indicated concentrations. After 4 hours at 37°C, supernatants were harvested and assayed for RANTES, IFNγ and MIP-1β content by ELISA (only RANTES shown). D. 2×10 3 868 CTL were incubated for 4 hours at 37°C with 1μg/ml of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV in an IFNγ ELISpot assay. E. 1.25×10 5 868 CTL were incubated with 1μg/ml of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV for 4 hours at 37°C. The supernatant was subsequently assayed for MIP-1β content by ELISA. Figures ( C-E ) show the mean ± SD of two replicate assays. Results similar to ( A-E ) were also obtained with tetramers conjugated to fluorochromes other than PE (data not shown). F. 003 CTL were incubated with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 GLCTLVAML or A2/K b GLCTLVAML at the indicated concentrations for 4 hours at 37°C, then stained with APC-conjugated anti-CD8 for 20 minutes on ice and assayed for CD107a mobilization as described in the Materials and Methods. The inset plot shows staining for APC-conjugated anti-CD8 on the x-axis and PE-conjugated A2/K b GLCTLVAML tetramer (5μg/ml) on the y-axis. Back-gated tetramer + CD107a + cells are shown in black and tetramer + CD107a − cells are shown in grey. Tetramer high CD8 high cells are preferentially activated by the A2/K b tetramer.
Figure Legend Snippet: A2/K b tetramers can activate CTL in the absence of a specific TCR/pMHCI interaction A. 10 5 003 CTL were suspended in 20μl PBS and stained with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV at the indicated concentrations and 7-AAD for 20 minutes at 37°C. Cells were then washed twice and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software. B. 10 5 003 CTL were suspended in 40μl R2 with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV at the indicated concentrations for 30 minutes at 37°C. Cells were subsequently stained with FITC-conjugated anti-αβ-TCR, 7-AAD and APC-conjugated anti-CD8 for 30 minutes on ice in azide buffer (0.1% azide/2% FCS/PBS). After two washes, data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software. C. 5×10 5 003 CTL were incubated with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV at the indicated concentrations. After 4 hours at 37°C, supernatants were harvested and assayed for RANTES, IFNγ and MIP-1β content by ELISA (only RANTES shown). D. 2×10 3 868 CTL were incubated for 4 hours at 37°C with 1μg/ml of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV in an IFNγ ELISpot assay. E. 1.25×10 5 868 CTL were incubated with 1μg/ml of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV for 4 hours at 37°C. The supernatant was subsequently assayed for MIP-1β content by ELISA. Figures ( C-E ) show the mean ± SD of two replicate assays. Results similar to ( A-E ) were also obtained with tetramers conjugated to fluorochromes other than PE (data not shown). F. 003 CTL were incubated with the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 GLCTLVAML or A2/K b GLCTLVAML at the indicated concentrations for 4 hours at 37°C, then stained with APC-conjugated anti-CD8 for 20 minutes on ice and assayed for CD107a mobilization as described in the Materials and Methods. The inset plot shows staining for APC-conjugated anti-CD8 on the x-axis and PE-conjugated A2/K b GLCTLVAML tetramer (5μg/ml) on the y-axis. Back-gated tetramer + CD107a + cells are shown in black and tetramer + CD107a − cells are shown in grey. Tetramer high CD8 high cells are preferentially activated by the A2/K b tetramer.

Techniques Used: CTL Assay, Staining, Flow Cytometry, Cytometry, Software, Incubation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Cell surface-expressed A2/K b primes non-specific expansion of CD8 + cells 10 6 A2 + PBMC were incubated with 2×10 5 irradiated A2 D227K/T228A, A2 or A2/K b C1R cells that had previously been pulsed with 1μM ELAGIGILTV (Melan-A 26-35 ) peptide in R10. From day 3, IL-2 was added in increments to reach a maximum concentration of 200 IU/ml by day 10. Lines were subsequently stained with PE-conjugated A2 ELAGIGILTV tetramer followed by APC-conjugated anti-CD8 and 7-AAD. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.
Figure Legend Snippet: Cell surface-expressed A2/K b primes non-specific expansion of CD8 + cells 10 6 A2 + PBMC were incubated with 2×10 5 irradiated A2 D227K/T228A, A2 or A2/K b C1R cells that had previously been pulsed with 1μM ELAGIGILTV (Melan-A 26-35 ) peptide in R10. From day 3, IL-2 was added in increments to reach a maximum concentration of 200 IU/ml by day 10. Lines were subsequently stained with PE-conjugated A2 ELAGIGILTV tetramer followed by APC-conjugated anti-CD8 and 7-AAD. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software.

Techniques Used: Incubation, Irradiation, Concentration Assay, Staining, Flow Cytometry, Cytometry, Software

The exquisite specificity of pMHCI tetramer staining is lost when the strength of the pMHCI/CD8 interaction is increased by ~15-fold A. The 003 or NT1 CTL clones (10 5 cells) or the 868 CTL line (2.5×10 5 cells), all specific for HIV-1 p17 Gag 77-85 , were stained with 1μg of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV in 20μl PBS for 20 minutes at 37°C. Cells were then stained with APC-conjugated anti-CD8 and 7-AAD for 30 minutes on ice, washed twice and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software. B. 2.5×10 5 PBMC were suspended in 250 μl FACS buffer (2% FCS/PBS), then stained with 1μg of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV for 20 minutes at 37°C. Each sample was subsequently stained with APC-conjugated anti-CD8, PerCP-conjugated anti-CD3 and 7-AAD for 30 minutes on ice, washed twice and resuspended in FACS buffer. Data were acquired using a FACSCalibur flow cytometer and analysed with CellQuest software by gating on the live CD3 + population. The values shown represent the percent of CD3 + CD8 + cells that stain with the indicated tetramer.
Figure Legend Snippet: The exquisite specificity of pMHCI tetramer staining is lost when the strength of the pMHCI/CD8 interaction is increased by ~15-fold A. The 003 or NT1 CTL clones (10 5 cells) or the 868 CTL line (2.5×10 5 cells), all specific for HIV-1 p17 Gag 77-85 , were stained with 1μg of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV in 20μl PBS for 20 minutes at 37°C. Cells were then stained with APC-conjugated anti-CD8 and 7-AAD for 30 minutes on ice, washed twice and resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software. B. 2.5×10 5 PBMC were suspended in 250 μl FACS buffer (2% FCS/PBS), then stained with 1μg of the PE-conjugated tetramers A2 SLYNTVATL, A2/K b SLYNTVATL, A2 LLFGYPVYV or A2/K b LLFGYPVYV for 20 minutes at 37°C. Each sample was subsequently stained with APC-conjugated anti-CD8, PerCP-conjugated anti-CD3 and 7-AAD for 30 minutes on ice, washed twice and resuspended in FACS buffer. Data were acquired using a FACSCalibur flow cytometer and analysed with CellQuest software by gating on the live CD3 + population. The values shown represent the percent of CD3 + CD8 + cells that stain with the indicated tetramer.

Techniques Used: Staining, CTL Assay, Clone Assay, Flow Cytometry, Cytometry, Software, FACS

7) Product Images from "Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region"

Article Title: Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region

Journal: European Journal of Immunology

doi: 10.1002/eji.200636765

HLA-A2 and GFP-HLA-A2 vectors used in this study and cell surface expression of HLA-A2. (A) HLA-A2 and HLA-A2 mutants were expressed as full-length molecules in pCDNA3.1 (Invitrogen) under G418 (Neo) selection. (B) HLA-A2-GFP fusion constructs were expressed in pGFP-N2 (Perkin Elmer) under Zeo selection. Vectors were linearized by cutting with BglII or PvuII (highlighted) prior to transfection. (C) C1R cells were transfected with wild-type HLA-A2, DT227/8KA HLA-A2 or Q115E HLA-A2; vectors are shown in (A). Transfectants were grown up from single clones and clones with similar expression levels of HLA-A2 were selected. Cells (2.5×10 5 ) in 100 μL of FACS buffer were stained with 2.5 μL of the HLA-A2-specific antibody BB7.2 conjugated with FITC (Serotec) for 20 min, washed twice in FACS buffer and analysed on a FACSCalibur (BD Biosciences). Bars show average mean fluorescence intensity of two experiments performed 2 wk apart. (D) C1R targets were transfected with HLA-A2-GFP fusion constructs in the vector shown in (B) and cloned by limiting dilution. Clones were analysed for GFP expression by FACS. Mean fluorescence is shown for clones used as targets in activation assays.
Figure Legend Snippet: HLA-A2 and GFP-HLA-A2 vectors used in this study and cell surface expression of HLA-A2. (A) HLA-A2 and HLA-A2 mutants were expressed as full-length molecules in pCDNA3.1 (Invitrogen) under G418 (Neo) selection. (B) HLA-A2-GFP fusion constructs were expressed in pGFP-N2 (Perkin Elmer) under Zeo selection. Vectors were linearized by cutting with BglII or PvuII (highlighted) prior to transfection. (C) C1R cells were transfected with wild-type HLA-A2, DT227/8KA HLA-A2 or Q115E HLA-A2; vectors are shown in (A). Transfectants were grown up from single clones and clones with similar expression levels of HLA-A2 were selected. Cells (2.5×10 5 ) in 100 μL of FACS buffer were stained with 2.5 μL of the HLA-A2-specific antibody BB7.2 conjugated with FITC (Serotec) for 20 min, washed twice in FACS buffer and analysed on a FACSCalibur (BD Biosciences). Bars show average mean fluorescence intensity of two experiments performed 2 wk apart. (D) C1R targets were transfected with HLA-A2-GFP fusion constructs in the vector shown in (B) and cloned by limiting dilution. Clones were analysed for GFP expression by FACS. Mean fluorescence is shown for clones used as targets in activation assays.

Techniques Used: Expressing, Selection, Construct, Transfection, Clone Assay, FACS, Staining, Fluorescence, Plasmid Preparation, Activation Assay

8) Product Images from "Protective Regulatory T Cell Immune Response Induced by Intranasal Immunization With the Live-Attenuated Pneumococcal Vaccine SPY1 via the Transforming Growth Factor-β1-Smad2/3 Pathway"

Article Title: Protective Regulatory T Cell Immune Response Induced by Intranasal Immunization With the Live-Attenuated Pneumococcal Vaccine SPY1 via the Transforming Growth Factor-β1-Smad2/3 Pathway

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01754

SPY1 vaccination activates the expression of regulatory T cells (Tregs) molecule Foxp3. (A) On day 7 after the last immunization, mice lungs were aseptically removed and homogenized, and single lung cell was stained with anti-mouse CD4-FITC and anti-mouse CD25-APC, followed by anti-mouse Foxp3-APC according to the manufacturer’s instructions. Cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer then. The percentages of CD4 + T cells which were CD25 + Foxp3 + Treg were calculated. (B) On day 7 after the last immunization, mice lungs were aseptically removed, and total RNA were extracted. Expression of foxp3 mRNA was analyzed by quantitative real-time PCR. Data were shown as mean ± SD from three independent experiments. (C) On day 7 after the last immunization, mice lungs were aseptically removed, and lung sections were stained with anti-FOXP3 antibody followed by streptavidin horseradish peroxidase chemistry. Then, the sections were examined under light microscopy at 100× magnification. Scale bar = 100 µm. Images were representative of staining observed in the lungs of mice within the group ( n = 4–6 mice). * p
Figure Legend Snippet: SPY1 vaccination activates the expression of regulatory T cells (Tregs) molecule Foxp3. (A) On day 7 after the last immunization, mice lungs were aseptically removed and homogenized, and single lung cell was stained with anti-mouse CD4-FITC and anti-mouse CD25-APC, followed by anti-mouse Foxp3-APC according to the manufacturer’s instructions. Cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer then. The percentages of CD4 + T cells which were CD25 + Foxp3 + Treg were calculated. (B) On day 7 after the last immunization, mice lungs were aseptically removed, and total RNA were extracted. Expression of foxp3 mRNA was analyzed by quantitative real-time PCR. Data were shown as mean ± SD from three independent experiments. (C) On day 7 after the last immunization, mice lungs were aseptically removed, and lung sections were stained with anti-FOXP3 antibody followed by streptavidin horseradish peroxidase chemistry. Then, the sections were examined under light microscopy at 100× magnification. Scale bar = 100 µm. Images were representative of staining observed in the lungs of mice within the group ( n = 4–6 mice). * p

Techniques Used: Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Light Microscopy

9) Product Images from "Gene expression profile correlates with T cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy"

Article Title: Gene expression profile correlates with T cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-10-2563

Peripheral blood immune monitoring data. (A) PBMC’s from normal volunteers and DC trial patient pre-vaccination timepoints were thawed and stained for the expression of CD3, CD4 and CD25, followed by the intracellular labeling of Foxp3. Stained cells were acquired on a BD FacsCalibur flow cytometer and analyzed using FloJo software. The frequencies of CD3 + CD4 + Foxp3 + and CD3 + CD4 + CD25 + Foxp3 + PBMC’s between normal volunteers and glioblastoma patients enrolled in this trial are compared. (*p=0.04; **p=0.01) (B,C) Serum cytokine responses, measured pre- and day 14 post-vaccination, after the initial course of DC vaccination (B) or after booster DC vaccinations with either 5% imiquimod or poly ICLC (C). Serum from patients enrolled on this clinical trial was thawed, labeled with cytometric bead array (CBA) antibody-coated beads, washed and subjected to analysis on a BD FacsCalibur flow cytometer together with cytokine standards. Quantitative assessment of cytokine levels was accomplished with a Microsoft Excel-based CBA software program. (D) Th1/Th2 cytokine ratios. Raw cytokine data for serum TNF-α and IL-10 at each timepoint were divided to generate a Th1:Th2 ratio.
Figure Legend Snippet: Peripheral blood immune monitoring data. (A) PBMC’s from normal volunteers and DC trial patient pre-vaccination timepoints were thawed and stained for the expression of CD3, CD4 and CD25, followed by the intracellular labeling of Foxp3. Stained cells were acquired on a BD FacsCalibur flow cytometer and analyzed using FloJo software. The frequencies of CD3 + CD4 + Foxp3 + and CD3 + CD4 + CD25 + Foxp3 + PBMC’s between normal volunteers and glioblastoma patients enrolled in this trial are compared. (*p=0.04; **p=0.01) (B,C) Serum cytokine responses, measured pre- and day 14 post-vaccination, after the initial course of DC vaccination (B) or after booster DC vaccinations with either 5% imiquimod or poly ICLC (C). Serum from patients enrolled on this clinical trial was thawed, labeled with cytometric bead array (CBA) antibody-coated beads, washed and subjected to analysis on a BD FacsCalibur flow cytometer together with cytokine standards. Quantitative assessment of cytokine levels was accomplished with a Microsoft Excel-based CBA software program. (D) Th1/Th2 cytokine ratios. Raw cytokine data for serum TNF-α and IL-10 at each timepoint were divided to generate a Th1:Th2 ratio.

Techniques Used: Staining, Expressing, Labeling, Flow Cytometry, Cytometry, Software, Crocin Bleaching Assay

10) Product Images from "Anti-CD8 antibodies can trigger CD8+ T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining"

Article Title: Anti-CD8 antibodies can trigger CD8+ T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining

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

doi: 10.4049/jimmunol.1003941

Anti-human CD8 antibodies can either enhance or inhibit the binding of pMHCI tetramers 5×10 4 ILA1 ( A ), ALF3 ( B ), MEL5 ( C ) or MEL187.c5 ( D ) CD8 + T-cells were preincubated at 4°C for 25 minutes with each of the following individual anti-human CD8 antibodies in parallel: 100 μg/ml OKT8, 6.25 μg/ml SK1, 50 μg/ml MCD8, 100 μg/ml 32/M4, 100 μg/ml C8/144B (144B), 25 μg/ml DK25 and 100 μg/ml 2ST8.5H7 (CD8β). CD8 + T-cells were subsequently stained with cognate PE-conjugated HLA A*0201 tetramers (25 μg/ml) and 7-AAD as described in the Materials Methods. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software. Relative MFI values with respect to pMHCI tetramer staining in the absence of preincubation with anti-CD8 antibody are shown. Fluorescence in the absence of added cognate tetramer (con) is shown in each case. Data are representative of four separate experiments using ILA1 and ALF3 CD8 + T-cells, and six separate experiments using MEL5 and MEL187.c5 CD8 + T-cells.
Figure Legend Snippet: Anti-human CD8 antibodies can either enhance or inhibit the binding of pMHCI tetramers 5×10 4 ILA1 ( A ), ALF3 ( B ), MEL5 ( C ) or MEL187.c5 ( D ) CD8 + T-cells were preincubated at 4°C for 25 minutes with each of the following individual anti-human CD8 antibodies in parallel: 100 μg/ml OKT8, 6.25 μg/ml SK1, 50 μg/ml MCD8, 100 μg/ml 32/M4, 100 μg/ml C8/144B (144B), 25 μg/ml DK25 and 100 μg/ml 2ST8.5H7 (CD8β). CD8 + T-cells were subsequently stained with cognate PE-conjugated HLA A*0201 tetramers (25 μg/ml) and 7-AAD as described in the Materials Methods. Data were acquired using a FACSCalibur flow cytometer and analyzed with FlowJo software. Relative MFI values with respect to pMHCI tetramer staining in the absence of preincubation with anti-CD8 antibody are shown. Fluorescence in the absence of added cognate tetramer (con) is shown in each case. Data are representative of four separate experiments using ILA1 and ALF3 CD8 + T-cells, and six separate experiments using MEL5 and MEL187.c5 CD8 + T-cells.

Techniques Used: Binding Assay, Staining, Flow Cytometry, Cytometry, Software, Fluorescence

OKT8 increases TCR/pMHCI on-rates at the cell surface 5 × 10 5 ILA1 ( A ) or ALF3 ( B C) CD8 + T-cells were removed from culture, washed twice and resuspended in 100 μl PBS with or without 100 μg/ml OKT8 or 25 μg/ml DK25, then incubated at 4°C for 25 minutes. Cognate PE-conjugated HLA A*0201 tetramer was added in each case at 5 μg/ml. At various time points as indicated, 12 μl of cell suspension was removed and analyzed using a FACSCalibur flow cytometer with FlowJo software. In panel ( C ).
Figure Legend Snippet: OKT8 increases TCR/pMHCI on-rates at the cell surface 5 × 10 5 ILA1 ( A ) or ALF3 ( B C) CD8 + T-cells were removed from culture, washed twice and resuspended in 100 μl PBS with or without 100 μg/ml OKT8 or 25 μg/ml DK25, then incubated at 4°C for 25 minutes. Cognate PE-conjugated HLA A*0201 tetramer was added in each case at 5 μg/ml. At various time points as indicated, 12 μl of cell suspension was removed and analyzed using a FACSCalibur flow cytometer with FlowJo software. In panel ( C ).

Techniques Used: Incubation, Flow Cytometry, Cytometry, Software

11) Product Images from "Characterization of Envelope Glycoprotein Mutants for Human T-Cell Leukemia Virus Type 1 Infectivity and Immortalization"

Article Title: Characterization of Envelope Glycoprotein Mutants for Human T-Cell Leukemia Virus Type 1 Infectivity and Immortalization

Journal: Journal of Virology

doi: 10.1128/JVI.75.19.9553-9559.2001

Cell surface phenotypes of the ACH-envelope clone-immortalized cells. Immortalized cells were stained with anti-CD4 antibody–fluorescein isothiocyanate and anti-CD8 antibody–phycoerythrin (right panels) and isotype control antibodies (left panels) and analyzed with a FACScalibur flow cytometer. FL1-H and FL2-H indicate CD4 and CD8 levels, respectively.
Figure Legend Snippet: Cell surface phenotypes of the ACH-envelope clone-immortalized cells. Immortalized cells were stained with anti-CD4 antibody–fluorescein isothiocyanate and anti-CD8 antibody–phycoerythrin (right panels) and isotype control antibodies (left panels) and analyzed with a FACScalibur flow cytometer. FL1-H and FL2-H indicate CD4 and CD8 levels, respectively.

Techniques Used: Staining, Flow Cytometry, Cytometry

12) Product Images from "Combination of lenalidomide with vitamin D3 induces apoptosis in mantle cell lymphoma via demethylation of BIK"

Article Title: Combination of lenalidomide with vitamin D3 induces apoptosis in mantle cell lymphoma via demethylation of BIK

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.346

The combined Len/VD3 treatment activated caspase 9, induced mitochondrial depolarization and involved Bax. ( a ) The Len/VD3 treatment induced caspase 9 activation. MCL cells (2 × 10 5 cells/ml) were incubated for 4 days with or without 1 μ M Len, 100 nM VD3 or Len/VD3. Cells were then lysed and activation of caspase 9 was assessed by western blotting. A representative experiment out of three is shown. ( b ) The Len/VD3 treatment induced mitochondrial depolarization. Z-138 cells (2 × 10 5 cells/ml) were incubated for 4 days with or without 1 μ M Len and 100 nM VD3, and then stained with JC-1. A representative experiment out of three is shown. ( c – e ) Silencing of BAX prevented cell death induced by Len/VD3 combination. JEKO-1 cells (5 × 10 5 /ml) were seeded for 48 h with or without the Len/VD3 combination (1 μ M Len and 100 nM VD3) before transfection with sicontrol (siCt) or si BAX RNA. Then, transfected cells were reseeded (5 × 10 5 /ml) for additional 3 days with or without the Len/VD3 combination. ( c ) Western blotting analysis of Bax expression. Bax expression was assessed in siCt- and si BAX- transfected JEKO-1 cells treated or not with Len/VD3 combination. ( d ) Len/VD3 induced a Bax-dependent decrease in cellularity. The cellular density was measured by a direct counting. The data represent five independent experiments. ( e ) Len/VD3 induced a Bax-dependent cell death. Cells were stained with Annexin V and fluorescence was analyzed on a FACSCalibur. The data represent five independent experiments. * P
Figure Legend Snippet: The combined Len/VD3 treatment activated caspase 9, induced mitochondrial depolarization and involved Bax. ( a ) The Len/VD3 treatment induced caspase 9 activation. MCL cells (2 × 10 5 cells/ml) were incubated for 4 days with or without 1 μ M Len, 100 nM VD3 or Len/VD3. Cells were then lysed and activation of caspase 9 was assessed by western blotting. A representative experiment out of three is shown. ( b ) The Len/VD3 treatment induced mitochondrial depolarization. Z-138 cells (2 × 10 5 cells/ml) were incubated for 4 days with or without 1 μ M Len and 100 nM VD3, and then stained with JC-1. A representative experiment out of three is shown. ( c – e ) Silencing of BAX prevented cell death induced by Len/VD3 combination. JEKO-1 cells (5 × 10 5 /ml) were seeded for 48 h with or without the Len/VD3 combination (1 μ M Len and 100 nM VD3) before transfection with sicontrol (siCt) or si BAX RNA. Then, transfected cells were reseeded (5 × 10 5 /ml) for additional 3 days with or without the Len/VD3 combination. ( c ) Western blotting analysis of Bax expression. Bax expression was assessed in siCt- and si BAX- transfected JEKO-1 cells treated or not with Len/VD3 combination. ( d ) Len/VD3 induced a Bax-dependent decrease in cellularity. The cellular density was measured by a direct counting. The data represent five independent experiments. ( e ) Len/VD3 induced a Bax-dependent cell death. Cells were stained with Annexin V and fluorescence was analyzed on a FACSCalibur. The data represent five independent experiments. * P

Techniques Used: Activation Assay, Incubation, Western Blot, Staining, Transfection, Expressing, Fluorescence

13) Product Images from "Cannabinoid WIN55, 212-2 induces cell cycle arrest and inhibits the proliferation and migration of human BEL7402 hepatocellular carcinoma cells"

Article Title: Cannabinoid WIN55, 212-2 induces cell cycle arrest and inhibits the proliferation and migration of human BEL7402 hepatocellular carcinoma cells

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2015.4477

Effects of WIN treatment on cell cycle distribution, cell proliferation and protein expression in BEL7402 hepatocellular carcinoma cells. (A) The effects of WIN treatment on cell cycle distribution were determined by flow cytometry in BEL7402 cells. The labeled cells were analyzed using a FACSCalibur flow cytometer, and the percentage of cells in the G0/G1 and S phases was calculated using ModFit LT software. The data presented are representative of a typical experiment repeated three times. (B) BEL7402 cell proliferation was inhibited following treatment with various concentrations of WIN for 24 h. Values are expressed as the mean ± standard deviation. * P
Figure Legend Snippet: Effects of WIN treatment on cell cycle distribution, cell proliferation and protein expression in BEL7402 hepatocellular carcinoma cells. (A) The effects of WIN treatment on cell cycle distribution were determined by flow cytometry in BEL7402 cells. The labeled cells were analyzed using a FACSCalibur flow cytometer, and the percentage of cells in the G0/G1 and S phases was calculated using ModFit LT software. The data presented are representative of a typical experiment repeated three times. (B) BEL7402 cell proliferation was inhibited following treatment with various concentrations of WIN for 24 h. Values are expressed as the mean ± standard deviation. * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Labeling, Software, Standard Deviation

14) Product Images from "Characterization of HPV18 E6-specific T cell responses and establishment of HPV18 E6-expressing tumor model"

Article Title: Characterization of HPV18 E6-specific T cell responses and establishment of HPV18 E6-expressing tumor model

Journal: Vaccine

doi: 10.1016/j.vaccine.2017.05.081

Characterization of MHC class I restriction element for HPV18 E6aa67-75 peptide recognized by CD8+ T cells after DNA vaccination in C57BL/6 mouse 5~8 weeks old female C57BL/6 mice were vaccinated with 30 μg/mouse of pcDNA3-HPV18-E6 DNA through IM injection followed by electroporation. The mice were boosted with the same regimen three times with 1-week interval. 7 days after the last vaccination, splenocytes were harvested from the mice, stimulated with either HPV18 E6aa67-75 peptide (1 μg/ml), or irradiated HPV18 E6aa67-75 peptide loaded C1R-D b , or C1R-K b cells at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then collected, washed with PBS+0.5% BSA, surface stained with PE-conjugated anti-mouse CD8a antibody. After wash, the cells were then permeabilized, fixed and intracellularly stained with FITC-conjugated anti-mouse IFN-γ antibody. The cells were acquired with FACSCalibur flow cytometer and analyzed with CellQuest software. A. Representative flow cytometry data. B. Bar graph summary of the flow cytometry data. p- values were calculated by 2-tailed Student’s t test. ** = p
Figure Legend Snippet: Characterization of MHC class I restriction element for HPV18 E6aa67-75 peptide recognized by CD8+ T cells after DNA vaccination in C57BL/6 mouse 5~8 weeks old female C57BL/6 mice were vaccinated with 30 μg/mouse of pcDNA3-HPV18-E6 DNA through IM injection followed by electroporation. The mice were boosted with the same regimen three times with 1-week interval. 7 days after the last vaccination, splenocytes were harvested from the mice, stimulated with either HPV18 E6aa67-75 peptide (1 μg/ml), or irradiated HPV18 E6aa67-75 peptide loaded C1R-D b , or C1R-K b cells at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then collected, washed with PBS+0.5% BSA, surface stained with PE-conjugated anti-mouse CD8a antibody. After wash, the cells were then permeabilized, fixed and intracellularly stained with FITC-conjugated anti-mouse IFN-γ antibody. The cells were acquired with FACSCalibur flow cytometer and analyzed with CellQuest software. A. Representative flow cytometry data. B. Bar graph summary of the flow cytometry data. p- values were calculated by 2-tailed Student’s t test. ** = p

Techniques Used: Mouse Assay, Injection, Electroporation, Irradiation, Staining, Flow Cytometry, Cytometry, Software

Characterization of MHC class I restriction element for HPV18 E6aa67-75 peptide recognized by CD8+ T cells after DNA vaccination in HLA-A*0201 transgenic C57BL/6 mouse 5~8 weeks old female HLA-A*0201 transgenic C57BL/6 mice were vaccinated with 50 μg/mouse of pcDNA3-HPV18-E6 DNA through IM injection followed by electroporation. The mice were boosted with the same regimen three times with 4-day interval. 7 days after the last vaccination, splenocytes were harvested from the mice, stimulated with either HPV18 E6aa67-75 peptide (1 μg/ml), or irradiated HPV18 E6aa67-75 peptide loaded C1R, or C1R-AAD, or C1R-D b , or C1R-K b cells at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then collected, washed with PBS+0.5% BSA, surface stained with PE-conjugated anti-mouse CD8a antibody. After wash, the cells were then permeabilized, fixed and intracellularly stained with FITC-conjugated anti-mouse IFN-γ antibody. The cells were acquired with FACSCalibur flow cytometer and analyzed with CellQuest software. A. Representative flow cytometry data. B. Bar graph summary of the flow cytometry data. Data are represented as mean ± SD. p- values were calculated by 2-tailed Student’s t test. * = p
Figure Legend Snippet: Characterization of MHC class I restriction element for HPV18 E6aa67-75 peptide recognized by CD8+ T cells after DNA vaccination in HLA-A*0201 transgenic C57BL/6 mouse 5~8 weeks old female HLA-A*0201 transgenic C57BL/6 mice were vaccinated with 50 μg/mouse of pcDNA3-HPV18-E6 DNA through IM injection followed by electroporation. The mice were boosted with the same regimen three times with 4-day interval. 7 days after the last vaccination, splenocytes were harvested from the mice, stimulated with either HPV18 E6aa67-75 peptide (1 μg/ml), or irradiated HPV18 E6aa67-75 peptide loaded C1R, or C1R-AAD, or C1R-D b , or C1R-K b cells at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then collected, washed with PBS+0.5% BSA, surface stained with PE-conjugated anti-mouse CD8a antibody. After wash, the cells were then permeabilized, fixed and intracellularly stained with FITC-conjugated anti-mouse IFN-γ antibody. The cells were acquired with FACSCalibur flow cytometer and analyzed with CellQuest software. A. Representative flow cytometry data. B. Bar graph summary of the flow cytometry data. Data are represented as mean ± SD. p- values were calculated by 2-tailed Student’s t test. * = p

Techniques Used: Transgenic Assay, Mouse Assay, Injection, Electroporation, Irradiation, Staining, Flow Cytometry, Cytometry, Software

Characterization of HPV18-E6 peptide-specific CD8+ T cell responses after DNA vaccination A. Schema of the experiment. Briefly, 5~8 weeks old female C57BL/6 mice (3 mice/group) were vaccinated with 50 μg/mouse of pcDNA3-HPV18-E6 or empty pcDNA3 vector DNA through IM injection followed by electroporation. The mice were boosted twice with the same regimen with 4-day intervals. 7 days after the last vaccination, splenocytes were harvested from the mice, stimulated with HPV18 E6aa67-75 peptide (1 μg/ml) at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then harvested, washed with PBS+0.5% BSA, surface stained with PE-conjugated anti-mouse CD8a antibody. After wash, the cells were then permeabilized, fixed and intracellularly stained with FITC-conjugated anti-mouse IFN-γ antibody. The cells were acquired with FACSCalibur flow cytometer and analyzed with CellQuest software. B. Representative flow cytometry data. C. Bar graph summary of the flow cytometry data. Data are represented as mean ± SD. p- values were calculated by 2-tailed Student’s t test. * = p
Figure Legend Snippet: Characterization of HPV18-E6 peptide-specific CD8+ T cell responses after DNA vaccination A. Schema of the experiment. Briefly, 5~8 weeks old female C57BL/6 mice (3 mice/group) were vaccinated with 50 μg/mouse of pcDNA3-HPV18-E6 or empty pcDNA3 vector DNA through IM injection followed by electroporation. The mice were boosted twice with the same regimen with 4-day intervals. 7 days after the last vaccination, splenocytes were harvested from the mice, stimulated with HPV18 E6aa67-75 peptide (1 μg/ml) at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then harvested, washed with PBS+0.5% BSA, surface stained with PE-conjugated anti-mouse CD8a antibody. After wash, the cells were then permeabilized, fixed and intracellularly stained with FITC-conjugated anti-mouse IFN-γ antibody. The cells were acquired with FACSCalibur flow cytometer and analyzed with CellQuest software. B. Representative flow cytometry data. C. Bar graph summary of the flow cytometry data. Data are represented as mean ± SD. p- values were calculated by 2-tailed Student’s t test. * = p

Techniques Used: Mouse Assay, Plasmid Preparation, Injection, Electroporation, Staining, Flow Cytometry, Cytometry, Software

15) Product Images from "Myeloid-derived macrophages and secreted HSP90α induce pancreatic ductal adenocarcinoma development"

Article Title: Myeloid-derived macrophages and secreted HSP90α induce pancreatic ductal adenocarcinoma development

Journal: Oncoimmunology

doi: 10.1080/2162402X.2018.1424612

eHSP90α had a stimulatory effect on HPDE cell anchorage independence but not cell proliferation. (A) Growth curves of HPDE cells treated with control medium (CTRL) or MϕCM for 1 to 5 days in the presence of control IgG or anti-CD91 antibody. Trypan blue exclusion assay was performed to count the numbers of viable cells. The data represent the averages of three independent experiments, and each experiment was performed in triplicate. (B) Growth curves of HPDE cells treated with PBS or 15 μg/ml of rHSP90α for 1 to 5 days. Trypan blue exclusion assay was performed, and the averages of three independent experiments are shown. (C) Flow cytometric analyses of the cell-cycle phase distribution of control medium (CTRL)- vs . MϕCM-treated HPDE cells (upper panel) and PBS- vs . rHSP90α-treated HPDE cells (lower panel). HPDE cells were treated for 24 h and trypsinized for ethanol fixation and propidium iodide staining. The histograph of cell-cycle phases was obtained from 10000 cells analyzed using a FACSCalibur flow cytometer. (D) The levels of cyclin D1, CDK4, Ser-780-phosphorylated Rb, Rb, cyclin E, cyclin A, Tyr-15-phosphorylated CDK2, CDK2, cyclin B, Tyr-15-phosphorylated CDC2, CDC2, and GAPDH in HPDE cells treated with PBS, 15 μg/ml rHSP90α, or 15 μg/ml rHSP90α plus preimmune IgG or anti-CD91 antibody. (E) Soft-agar colony-forming assay of PBS or rHSP90α-treated HPDE cells. HPDE cells (2000 cells seeded on the soft-agar layer in each well of a 6-well plate) were treated with or without 15 μg/ml rHSP90α and refreshed every 3 days for 3 weeks. The colonies were stained with Giemsa and photographed under a microscope (100 ×); only colonies consisting of ≥80 cells were counted. The mean ± SD values of three independent experiments are shown, and each experiment was performed in triplicate. # , P
Figure Legend Snippet: eHSP90α had a stimulatory effect on HPDE cell anchorage independence but not cell proliferation. (A) Growth curves of HPDE cells treated with control medium (CTRL) or MϕCM for 1 to 5 days in the presence of control IgG or anti-CD91 antibody. Trypan blue exclusion assay was performed to count the numbers of viable cells. The data represent the averages of three independent experiments, and each experiment was performed in triplicate. (B) Growth curves of HPDE cells treated with PBS or 15 μg/ml of rHSP90α for 1 to 5 days. Trypan blue exclusion assay was performed, and the averages of three independent experiments are shown. (C) Flow cytometric analyses of the cell-cycle phase distribution of control medium (CTRL)- vs . MϕCM-treated HPDE cells (upper panel) and PBS- vs . rHSP90α-treated HPDE cells (lower panel). HPDE cells were treated for 24 h and trypsinized for ethanol fixation and propidium iodide staining. The histograph of cell-cycle phases was obtained from 10000 cells analyzed using a FACSCalibur flow cytometer. (D) The levels of cyclin D1, CDK4, Ser-780-phosphorylated Rb, Rb, cyclin E, cyclin A, Tyr-15-phosphorylated CDK2, CDK2, cyclin B, Tyr-15-phosphorylated CDC2, CDC2, and GAPDH in HPDE cells treated with PBS, 15 μg/ml rHSP90α, or 15 μg/ml rHSP90α plus preimmune IgG or anti-CD91 antibody. (E) Soft-agar colony-forming assay of PBS or rHSP90α-treated HPDE cells. HPDE cells (2000 cells seeded on the soft-agar layer in each well of a 6-well plate) were treated with or without 15 μg/ml rHSP90α and refreshed every 3 days for 3 weeks. The colonies were stained with Giemsa and photographed under a microscope (100 ×); only colonies consisting of ≥80 cells were counted. The mean ± SD values of three independent experiments are shown, and each experiment was performed in triplicate. # , P

Techniques Used: Trypan Blue Exclusion Assay, Flow Cytometry, Staining, Cytometry, Microscopy

16) Product Images from "Targeting of the Yersinia pestis F1 capsular antigen by innate-like B1b cells mediates a rapid protective response against bubonic plague"

Article Title: Targeting of the Yersinia pestis F1 capsular antigen by innate-like B1b cells mediates a rapid protective response against bubonic plague

Journal: NPJ Vaccines

doi: 10.1038/s41541-018-0087-z

F1 is associated with peritoneal cells shortly after vaccination. Representative dot plot analysis of gated cells isolated from the draining inguinal lymph node (ILN), spleen and peritoneal cavity (PerC) of mice ( n = 6) vaccinated with labeled F1 (polymeric form, 80 μg/mouse, upper panel). Control mice were vaccinated with non-labeled F1 ( n = 3, lower panel). FL1 and FL4 indicate fluorescence detected through 530/30 nm and 661/16 nm filters of a FACSCalibur™ flow cytometer (BD Bioscience), respectively. Quadrant lines mark negatively and positively stained cells
Figure Legend Snippet: F1 is associated with peritoneal cells shortly after vaccination. Representative dot plot analysis of gated cells isolated from the draining inguinal lymph node (ILN), spleen and peritoneal cavity (PerC) of mice ( n = 6) vaccinated with labeled F1 (polymeric form, 80 μg/mouse, upper panel). Control mice were vaccinated with non-labeled F1 ( n = 3, lower panel). FL1 and FL4 indicate fluorescence detected through 530/30 nm and 661/16 nm filters of a FACSCalibur™ flow cytometer (BD Bioscience), respectively. Quadrant lines mark negatively and positively stained cells

Techniques Used: Isolation, Mouse Assay, Labeling, Fluorescence, Flow Cytometry, Cytometry, Staining

17) Product Images from "Protective Regulatory T Cell Immune Response Induced by Intranasal Immunization With the Live-Attenuated Pneumococcal Vaccine SPY1 via the Transforming Growth Factor-β1-Smad2/3 Pathway"

Article Title: Protective Regulatory T Cell Immune Response Induced by Intranasal Immunization With the Live-Attenuated Pneumococcal Vaccine SPY1 via the Transforming Growth Factor-β1-Smad2/3 Pathway

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01754

SPY1 vaccination activates the expression of regulatory T cells (Tregs) molecule Foxp3. (A) On day 7 after the last immunization, mice lungs were aseptically removed and homogenized, and single lung cell was stained with anti-mouse CD4-FITC and anti-mouse CD25-APC, followed by anti-mouse Foxp3-APC according to the manufacturer’s instructions. Cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer then. The percentages of CD4 + T cells which were CD25 + Foxp3 + Treg were calculated. (B) On day 7 after the last immunization, mice lungs were aseptically removed, and total RNA were extracted. Expression of foxp3 mRNA was analyzed by quantitative real-time PCR. Data were shown as mean ± SD from three independent experiments. (C) On day 7 after the last immunization, mice lungs were aseptically removed, and lung sections were stained with anti-FOXP3 antibody followed by streptavidin horseradish peroxidase chemistry. Then, the sections were examined under light microscopy at 100× magnification. Scale bar = 100 µm. Images were representative of staining observed in the lungs of mice within the group ( n = 4–6 mice). * p
Figure Legend Snippet: SPY1 vaccination activates the expression of regulatory T cells (Tregs) molecule Foxp3. (A) On day 7 after the last immunization, mice lungs were aseptically removed and homogenized, and single lung cell was stained with anti-mouse CD4-FITC and anti-mouse CD25-APC, followed by anti-mouse Foxp3-APC according to the manufacturer’s instructions. Cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer then. The percentages of CD4 + T cells which were CD25 + Foxp3 + Treg were calculated. (B) On day 7 after the last immunization, mice lungs were aseptically removed, and total RNA were extracted. Expression of foxp3 mRNA was analyzed by quantitative real-time PCR. Data were shown as mean ± SD from three independent experiments. (C) On day 7 after the last immunization, mice lungs were aseptically removed, and lung sections were stained with anti-FOXP3 antibody followed by streptavidin horseradish peroxidase chemistry. Then, the sections were examined under light microscopy at 100× magnification. Scale bar = 100 µm. Images were representative of staining observed in the lungs of mice within the group ( n = 4–6 mice). * p

Techniques Used: Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Light Microscopy

18) Product Images from "Huachansu mediates cell death in non-Hodgkin’s lymphoma by induction of caspase-3 and inhibition of MAP kinase"

Article Title: Huachansu mediates cell death in non-Hodgkin’s lymphoma by induction of caspase-3 and inhibition of MAP kinase

Journal: International Journal of Oncology

doi: 10.3892/ijo.2015.3044

HCS triggers cell arrest and induces cell death of Ramos cells. Cells were treated with HCS for 24 h. (A) Histograms of Ramos cells treated with vehicle (a), HCS 5 μl/ml (b) and HCS 25 μl/ml (c) and quantitative analysis of the cell cycle alteration elicited by HCS (d). (B) Cells were treated with HCS at similar concentrations illustrated in sub-figure A, and apoptotic cells were assayed by flow cytometry following Annexin V staining, using a FACSCalibur instrument. Each experiment was performed in duplicate and repeated twice independently. Each bar graph represents the mean and the error bars represent ± SEM. * P
Figure Legend Snippet: HCS triggers cell arrest and induces cell death of Ramos cells. Cells were treated with HCS for 24 h. (A) Histograms of Ramos cells treated with vehicle (a), HCS 5 μl/ml (b) and HCS 25 μl/ml (c) and quantitative analysis of the cell cycle alteration elicited by HCS (d). (B) Cells were treated with HCS at similar concentrations illustrated in sub-figure A, and apoptotic cells were assayed by flow cytometry following Annexin V staining, using a FACSCalibur instrument. Each experiment was performed in duplicate and repeated twice independently. Each bar graph represents the mean and the error bars represent ± SEM. * P

Techniques Used: Flow Cytometry, Cytometry, Staining

19) Product Images from "Cord blood stem cells revert glioma stem cell EMT by down regulating transcriptional activation of Sox2 and Twist1"

Article Title: Cord blood stem cells revert glioma stem cell EMT by down regulating transcriptional activation of Sox2 and Twist1

Journal: Oncotarget

doi:

Effect of siTwist and siSox2 on Twist1 and Sox2 expression using frequency distribution contour plots Approximately 1×10 5 U251 (A), U87 (B), 4910 (C), and 5310 (D) cells were transfected with either siTwist or si-Sox2. Samples were harvested after 72 hrs. siTwist1 and siSox2 expression was determined by staining with the mAb to human Twist1, Sox2 or isotype control mouse or goat IgG 1 , and then followed by a secondary antibody goat anti-mouse IgG or donkey anti-goat IgG conjugated to Alexa Fluor red or green dye. The background was subtracted using different isotypic controls for respective antibodies. In overlays under Figures 4A-4D , purple color represents the negative control, green color indicates the siTwist or siSox2 treatment, orange color represents Sox2 expression, and red color indicates Twist1 expression. Bar graph (E) represents the expression level of Twist1 in siTwist1 knock down (F) represents the expression level of Sox2 in siSox2 knockdown (G) represents the expression level of Sox2 in siTwist1 knock down while (H) represents the percent expression levels of Twist1 in siSox2 knock down as quantified from the above contour plots. Cells were analyzed with a FACSCalibur flow cytometer and CellQuest software.
Figure Legend Snippet: Effect of siTwist and siSox2 on Twist1 and Sox2 expression using frequency distribution contour plots Approximately 1×10 5 U251 (A), U87 (B), 4910 (C), and 5310 (D) cells were transfected with either siTwist or si-Sox2. Samples were harvested after 72 hrs. siTwist1 and siSox2 expression was determined by staining with the mAb to human Twist1, Sox2 or isotype control mouse or goat IgG 1 , and then followed by a secondary antibody goat anti-mouse IgG or donkey anti-goat IgG conjugated to Alexa Fluor red or green dye. The background was subtracted using different isotypic controls for respective antibodies. In overlays under Figures 4A-4D , purple color represents the negative control, green color indicates the siTwist or siSox2 treatment, orange color represents Sox2 expression, and red color indicates Twist1 expression. Bar graph (E) represents the expression level of Twist1 in siTwist1 knock down (F) represents the expression level of Sox2 in siSox2 knockdown (G) represents the expression level of Sox2 in siTwist1 knock down while (H) represents the percent expression levels of Twist1 in siSox2 knock down as quantified from the above contour plots. Cells were analyzed with a FACSCalibur flow cytometer and CellQuest software.

Techniques Used: Expressing, Transfection, Staining, Negative Control, Flow Cytometry, Cytometry, Software

20) Product Images from "Cord blood stem cells revert glioma stem cell EMT by down regulating transcriptional activation of Sox2 and Twist1"

Article Title: Cord blood stem cells revert glioma stem cell EMT by down regulating transcriptional activation of Sox2 and Twist1

Journal: Oncotarget

doi:

Effect of siTwist and siSox2 on Twist1 and Sox2 expression using frequency distribution contour plots Approximately 1×10 5 U251 (A), U87 (B), 4910 (C), and 5310 (D) cells were transfected with either siTwist or si-Sox2. Samples were harvested after 72 hrs. siTwist1 and siSox2 expression was determined by staining with the mAb to human Twist1, Sox2 or isotype control mouse or goat IgG 1 , and then followed by a secondary antibody goat anti-mouse IgG or donkey anti-goat IgG conjugated to Alexa Fluor red or green dye. The background was subtracted using different isotypic controls for respective antibodies. In overlays under Figures 4A-4D , purple color represents the negative control, green color indicates the siTwist or siSox2 treatment, orange color represents Sox2 expression, and red color indicates Twist1 expression. Bar graph (E) represents the expression level of Twist1 in siTwist1 knock down (F) represents the expression level of Sox2 in siSox2 knockdown (G) represents the expression level of Sox2 in siTwist1 knock down while (H) represents the percent expression levels of Twist1 in siSox2 knock down as quantified from the above contour plots. Cells were analyzed with a FACSCalibur flow cytometer and CellQuest software.
Figure Legend Snippet: Effect of siTwist and siSox2 on Twist1 and Sox2 expression using frequency distribution contour plots Approximately 1×10 5 U251 (A), U87 (B), 4910 (C), and 5310 (D) cells were transfected with either siTwist or si-Sox2. Samples were harvested after 72 hrs. siTwist1 and siSox2 expression was determined by staining with the mAb to human Twist1, Sox2 or isotype control mouse or goat IgG 1 , and then followed by a secondary antibody goat anti-mouse IgG or donkey anti-goat IgG conjugated to Alexa Fluor red or green dye. The background was subtracted using different isotypic controls for respective antibodies. In overlays under Figures 4A-4D , purple color represents the negative control, green color indicates the siTwist or siSox2 treatment, orange color represents Sox2 expression, and red color indicates Twist1 expression. Bar graph (E) represents the expression level of Twist1 in siTwist1 knock down (F) represents the expression level of Sox2 in siSox2 knockdown (G) represents the expression level of Sox2 in siTwist1 knock down while (H) represents the percent expression levels of Twist1 in siSox2 knock down as quantified from the above contour plots. Cells were analyzed with a FACSCalibur flow cytometer and CellQuest software.

Techniques Used: Expressing, Transfection, Staining, Negative Control, Flow Cytometry, Cytometry, Software

21) Product Images from "Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III"

Article Title: Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00059

Antibodies from mice immunized with pscDEC-EDIII and pscISO-EDIII partially inhibit DENV infection by binding to EDIII. (A) Neutralization in VERO cells. Pooled sera from mice immunized as described in Figure 2 were heat inactivated at 56°C for 30 min. Two-fold serially diluted sera were incubated with the DENV2 particles for 30 min at 37°C and 5% CO 2 . The sera/virus mixture was then incubated with the cells for 1 h at 37°C and 5% CO 2 . The cultures supernatant was replaced by DMEM 5% FBS followed by another incubation at the same conditions for 24 h. Cells were stained with the 4G2 (mouse anti-flavivirus envelope antibody) and anti-mouse IgG-Alexa488. The neutralization of infection was determined in comparison to a control infection. Results are represented by means and SEM from pooled data of four independent experiments. (B) Competition assay with the recombinant EDIII protein. A fixed dilution (1:20) of sera was incubated with increasing molar concentrations of EDIII protein prior to incubation with the virus. The cells were resuspended in FACS buffer and 20,000 events were acquired in the BD FACSCalibur™ (BD biosciences) flow cytometer. The neutralization of infection was determined in comparison to a control infection with sera derived from mice injected with saline. Data were analyzed by a two-way ANOVA for repetitive measures followed by Sidak's multiple comparisons test. ns = not-significant. Representative of three independent experiments.
Figure Legend Snippet: Antibodies from mice immunized with pscDEC-EDIII and pscISO-EDIII partially inhibit DENV infection by binding to EDIII. (A) Neutralization in VERO cells. Pooled sera from mice immunized as described in Figure 2 were heat inactivated at 56°C for 30 min. Two-fold serially diluted sera were incubated with the DENV2 particles for 30 min at 37°C and 5% CO 2 . The sera/virus mixture was then incubated with the cells for 1 h at 37°C and 5% CO 2 . The cultures supernatant was replaced by DMEM 5% FBS followed by another incubation at the same conditions for 24 h. Cells were stained with the 4G2 (mouse anti-flavivirus envelope antibody) and anti-mouse IgG-Alexa488. The neutralization of infection was determined in comparison to a control infection. Results are represented by means and SEM from pooled data of four independent experiments. (B) Competition assay with the recombinant EDIII protein. A fixed dilution (1:20) of sera was incubated with increasing molar concentrations of EDIII protein prior to incubation with the virus. The cells were resuspended in FACS buffer and 20,000 events were acquired in the BD FACSCalibur™ (BD biosciences) flow cytometer. The neutralization of infection was determined in comparison to a control infection with sera derived from mice injected with saline. Data were analyzed by a two-way ANOVA for repetitive measures followed by Sidak's multiple comparisons test. ns = not-significant. Representative of three independent experiments.

Techniques Used: Mouse Assay, Infection, Binding Assay, Neutralization, Incubation, Staining, Competitive Binding Assay, Recombinant, FACS, Flow Cytometry, Cytometry, Derivative Assay, Injection

22) Product Images from "Regulation of Abro1/KIAA0157 during myocardial infarction and cell death reveals a novel cardioprotective mechanism for Lys63-specific deubiquitination"

Article Title: Regulation of Abro1/KIAA0157 during myocardial infarction and cell death reveals a novel cardioprotective mechanism for Lys63-specific deubiquitination

Journal: Journal of molecular and cellular cardiology

doi: 10.1016/j.yjmcc.2010.12.015

Abro1 overexpression protects H9c2 cells from H 2 O 2 induced apoptosis. H9c2 cells were transfected with empty GFP vector or GFP-Abro1 and apoptosis was induced using 100μM or 200μM of H 2 O 2 for 18 hours. Cells were stained with Annexin V and analyzed using a FACScalibur Flow Cytometer (BD Biosciences). Data are mean ± standard deviation of four independent experiments. ‡ P
Figure Legend Snippet: Abro1 overexpression protects H9c2 cells from H 2 O 2 induced apoptosis. H9c2 cells were transfected with empty GFP vector or GFP-Abro1 and apoptosis was induced using 100μM or 200μM of H 2 O 2 for 18 hours. Cells were stained with Annexin V and analyzed using a FACScalibur Flow Cytometer (BD Biosciences). Data are mean ± standard deviation of four independent experiments. ‡ P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Standard Deviation

23) Product Images from "PKCδ-Mediated Nox2 Activation Promotes Fluid-Phase Pinocytosis of Antigens by Immature Dendritic Cells"

Article Title: PKCδ-Mediated Nox2 Activation Promotes Fluid-Phase Pinocytosis of Antigens by Immature Dendritic Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00537

Protein kinase C activation promotes macropinocytosis of antigens in stimulated immature DCs (iDCs). (A) Wild-type (WT) bone marrow-derived immature dendritic cells (BMiDCs) were incubated with FITC-OVA (50 µg/mL; 45,000 MW) and treated with or without phorbol 12-myristate 13-acetate (PMA, 1 µM) for 5 h. Cells were stained with PE/Cy7-conjugated CD11c (or respective isotype control) monoclonal antibodies, fixed in 2% paraformaldehyde, and FITC fluorescence analyzed in PE/Cy7-gated CD11c + cells using a Becton Dickinson FACSCalibur flow cytometer. Gating strategy for flow cytometry analysis is shown in Figure S1 in Supplementary Material. Bar diagram shows the mean fold change in percentage of FITC fluorescence + cells among CD11c + population ( n = 7). (B) WT CD11c + BMiDC were incubated with FITC-OVA (50 µg/mL, green) and stimulated with vehicle or PMA (1 µM) for 5 h. Cells were fixed in 2% PFA, and stained with TRITC-phalloidin (red) and Hoechst 33342 (blue). Images were taken using a Zeiss 780 inverted confocal microscope (63×). Similar findings have been observed in three independent experiments. Scale bar: 5 µm. Bar diagram represents quantified mean FITC fluorescence normalized to cell area in vehicle- and PMA-treated cells. (C) WT CD11c + BMiDCs were treated with PMA (1 µM) for 30 min and incubated with the plasma membrane dye FM™ 4-64 (green) and Alexa Fluor 488-OVA (AF-OVA, 50 µg/mL, red) for 10 min. Live cell imaging was performed using a Zeiss 780 inverted confocal microscope. Yellow arrows indicate membrane ruffles, white arrows indicate membrane-derived macropinosomes, and blue arrows point to internalized extracellular solutes in macropinosomes. Images are representative of three independent experiments. Scale bar: 5 µm. (D) WT BMiDC were preincubated with vehicle, cytochalasin D (Cyto. D, 1 µM, 30 min) or LY294002 (10 µM, 30 min), treated with PMA (1 µM), and incubated with FITC-OVA for 5 h. FITC-OVA uptake was analyzed by fluorescence-activated cell sorting (FACS) ( n = 3). (E) WT BMiDC were preincubated with vehicle or EIPA (25 µM) for 30 min, and challenged with PMA (1 µM, 5 h) in the presence of FITC-OVA. FITC-OVA internalization was analyzed by FACS ( n = 5). (F,G) WT BMiDC were preincubated with vehicle or calphostin C (Calp. C, 100 nM, 30 min), treated with PMA, and FACS was performed as described above ( n = 3). (H) WT BMiDC were preincubated with vehicle or calphostin C (Calp. C, 100 nM, 30 min), treated with HGF (100 ng/mL) in the presence of FITC-OVA for 5 h and FACS was performed to analyze FITC-OVA internalization. Bar diagram represents the mean fold change in FITC fluorescence + cells ( n = 3). Data represent the mean ± SD. * p
Figure Legend Snippet: Protein kinase C activation promotes macropinocytosis of antigens in stimulated immature DCs (iDCs). (A) Wild-type (WT) bone marrow-derived immature dendritic cells (BMiDCs) were incubated with FITC-OVA (50 µg/mL; 45,000 MW) and treated with or without phorbol 12-myristate 13-acetate (PMA, 1 µM) for 5 h. Cells were stained with PE/Cy7-conjugated CD11c (or respective isotype control) monoclonal antibodies, fixed in 2% paraformaldehyde, and FITC fluorescence analyzed in PE/Cy7-gated CD11c + cells using a Becton Dickinson FACSCalibur flow cytometer. Gating strategy for flow cytometry analysis is shown in Figure S1 in Supplementary Material. Bar diagram shows the mean fold change in percentage of FITC fluorescence + cells among CD11c + population ( n = 7). (B) WT CD11c + BMiDC were incubated with FITC-OVA (50 µg/mL, green) and stimulated with vehicle or PMA (1 µM) for 5 h. Cells were fixed in 2% PFA, and stained with TRITC-phalloidin (red) and Hoechst 33342 (blue). Images were taken using a Zeiss 780 inverted confocal microscope (63×). Similar findings have been observed in three independent experiments. Scale bar: 5 µm. Bar diagram represents quantified mean FITC fluorescence normalized to cell area in vehicle- and PMA-treated cells. (C) WT CD11c + BMiDCs were treated with PMA (1 µM) for 30 min and incubated with the plasma membrane dye FM™ 4-64 (green) and Alexa Fluor 488-OVA (AF-OVA, 50 µg/mL, red) for 10 min. Live cell imaging was performed using a Zeiss 780 inverted confocal microscope. Yellow arrows indicate membrane ruffles, white arrows indicate membrane-derived macropinosomes, and blue arrows point to internalized extracellular solutes in macropinosomes. Images are representative of three independent experiments. Scale bar: 5 µm. (D) WT BMiDC were preincubated with vehicle, cytochalasin D (Cyto. D, 1 µM, 30 min) or LY294002 (10 µM, 30 min), treated with PMA (1 µM), and incubated with FITC-OVA for 5 h. FITC-OVA uptake was analyzed by fluorescence-activated cell sorting (FACS) ( n = 3). (E) WT BMiDC were preincubated with vehicle or EIPA (25 µM) for 30 min, and challenged with PMA (1 µM, 5 h) in the presence of FITC-OVA. FITC-OVA internalization was analyzed by FACS ( n = 5). (F,G) WT BMiDC were preincubated with vehicle or calphostin C (Calp. C, 100 nM, 30 min), treated with PMA, and FACS was performed as described above ( n = 3). (H) WT BMiDC were preincubated with vehicle or calphostin C (Calp. C, 100 nM, 30 min), treated with HGF (100 ng/mL) in the presence of FITC-OVA for 5 h and FACS was performed to analyze FITC-OVA internalization. Bar diagram represents the mean fold change in FITC fluorescence + cells ( n = 3). Data represent the mean ± SD. * p

Techniques Used: Activation Assay, Derivative Assay, Incubation, Staining, Fluorescence, Flow Cytometry, Cytometry, Microscopy, Live Cell Imaging, FACS

24) Product Images from "Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane"

Article Title: Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane

Journal: Viruses

doi: 10.3390/v11121140

Effect on the localization of gp51 by mutant forms of the infectious molecular clone pBLV-IF2. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pBLV-IF2, mutant pBLV-IF2, or control pBluescript II SK (−) using 8 μL of FuGENE HD. The transfection efficiency was similar among all mutant pBLV-IF2s as evaluated according to the ratio of GFP-expressing HeLa cells determined via FACSCalibur™ flow cytometry. ( A ) To detect cell surface gp51, cells were fixed and stained with anti-gp51 MAb, followed by Alexa Fluor 488-conjugated anti-Mouse IgG, then stained with Hoechst 33342 and observed using an FV-1000 fluorescence microscope (right panel). ( B ) To detect intracellular Env protein, cells were fixed, permeabilized with 0.5% Triton X-100, stained with anti-BLVgp51 MAb followed by Alexa Fluor 488-conjugated anti-Mouse IgG, and observed using an FV-1000 fluorescence microscope (right panel). ( A , B ) Fluorescence intensity maps were plotted for linear transects drawn through the nuclei by line scan measurements through each cell using FV10-ASW 4.02 microscope software, and fluorescence intensities on the cell surface were measured. The width of each line was thinner than 1 pixel. Peak membrane intensity was normalized by the mean intensity of pBLV-IF2 for each experiment. The results show the relative intensities of at least 50 cells expressing gp51 over seven independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p
Figure Legend Snippet: Effect on the localization of gp51 by mutant forms of the infectious molecular clone pBLV-IF2. HeLa cells (1.0 × 10 5 ) were seeded on a coverslip in a 12 well plate the day prior to transfection and transfected with 2 µg of either wild-type pBLV-IF2, mutant pBLV-IF2, or control pBluescript II SK (−) using 8 μL of FuGENE HD. The transfection efficiency was similar among all mutant pBLV-IF2s as evaluated according to the ratio of GFP-expressing HeLa cells determined via FACSCalibur™ flow cytometry. ( A ) To detect cell surface gp51, cells were fixed and stained with anti-gp51 MAb, followed by Alexa Fluor 488-conjugated anti-Mouse IgG, then stained with Hoechst 33342 and observed using an FV-1000 fluorescence microscope (right panel). ( B ) To detect intracellular Env protein, cells were fixed, permeabilized with 0.5% Triton X-100, stained with anti-BLVgp51 MAb followed by Alexa Fluor 488-conjugated anti-Mouse IgG, and observed using an FV-1000 fluorescence microscope (right panel). ( A , B ) Fluorescence intensity maps were plotted for linear transects drawn through the nuclei by line scan measurements through each cell using FV10-ASW 4.02 microscope software, and fluorescence intensities on the cell surface were measured. The width of each line was thinner than 1 pixel. Peak membrane intensity was normalized by the mean intensity of pBLV-IF2 for each experiment. The results show the relative intensities of at least 50 cells expressing gp51 over seven independent experiments. Each column and error bar represents the mean ± SD of intensity for all cells. All values were analyzed by two-way ANOVA with Dunnett’s test. The asterisk indicates a statistically significant difference (* p

Techniques Used: Mutagenesis, Transfection, Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence, Microscopy, Software

25) Product Images from "Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells"

Article Title: Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01405

Comparison of PTA and ZOL in the induction of IFN-γ from Vγ2Vδ2 T cells. (A) Determination of IFN-γ produced by Vγ2Vδ2 T cells in response to lung cancer cell lines pulsed with PTA or ZOL. Human lung cancer cells, PC-9, PC-6, H1975, and H520 (4 × 10 5 cells), pretreated with PTA (•) at concentrations of 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 nM, or ZOL (⚬) at concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 μM were challenged by PTA-expanded Vγ2Vδ2 T cells (4 × 10 5 cells). After incubation for 16 h, the culture supernatants were examined for IFN-γ levels through ELISA. (B) Intracellular staining of IFN-γ in Vγ2Vδ2 T cells in response to human lung cancer cells pretreated with PTA or ZOL. PC-9 human lung cancer cells (1 × 10 6 cells/ml) were treated with 1 ml of the complete RPMI1640 medium or the medium containing 1 μM PTA or with 1 mM ZOL at 37°C with 5% CO 2 for 2 h. Then, the cells were examined for intracellular IFN-γ using a FACSCalibur flow cytometer and the cell population was visualized using FlowJo ver. 10.
Figure Legend Snippet: Comparison of PTA and ZOL in the induction of IFN-γ from Vγ2Vδ2 T cells. (A) Determination of IFN-γ produced by Vγ2Vδ2 T cells in response to lung cancer cell lines pulsed with PTA or ZOL. Human lung cancer cells, PC-9, PC-6, H1975, and H520 (4 × 10 5 cells), pretreated with PTA (•) at concentrations of 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 nM, or ZOL (⚬) at concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, or 1,000 μM were challenged by PTA-expanded Vγ2Vδ2 T cells (4 × 10 5 cells). After incubation for 16 h, the culture supernatants were examined for IFN-γ levels through ELISA. (B) Intracellular staining of IFN-γ in Vγ2Vδ2 T cells in response to human lung cancer cells pretreated with PTA or ZOL. PC-9 human lung cancer cells (1 × 10 6 cells/ml) were treated with 1 ml of the complete RPMI1640 medium or the medium containing 1 μM PTA or with 1 mM ZOL at 37°C with 5% CO 2 for 2 h. Then, the cells were examined for intracellular IFN-γ using a FACSCalibur flow cytometer and the cell population was visualized using FlowJo ver. 10.

Techniques Used: Produced, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

26) Product Images from "Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin Bound-Paclitaxel in Pancreatic Ductal Adenocarcinoma"

Article Title: Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin Bound-Paclitaxel in Pancreatic Ductal Adenocarcinoma

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-14-0028

Pam inhibits the proliferation and induces the apoptosis and cell cycle arrest of PSCs. (A) Cell viability was determined using the MTS cell proliferation assay. The results are expressed as a percentage of growth inhibition with respect to untreated cells. Error bars represent the standard deviation (SD) of the mean of triplicates. PSCs were analyzed in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) to determine the number of apoptotic cells (B). Western blotting results for cleaved caspase-3 and cleaved PARP-1 (C). Distribution of cells in each phase of the cell cycle (D). Western blotting for p27 and cdk2 (E); *, P
Figure Legend Snippet: Pam inhibits the proliferation and induces the apoptosis and cell cycle arrest of PSCs. (A) Cell viability was determined using the MTS cell proliferation assay. The results are expressed as a percentage of growth inhibition with respect to untreated cells. Error bars represent the standard deviation (SD) of the mean of triplicates. PSCs were analyzed in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) to determine the number of apoptotic cells (B). Western blotting results for cleaved caspase-3 and cleaved PARP-1 (C). Distribution of cells in each phase of the cell cycle (D). Western blotting for p27 and cdk2 (E); *, P

Techniques Used: Proliferation Assay, Inhibition, Standard Deviation, Flow Cytometry, Cytometry, Western Blot

27) Product Images from "Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes 1Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes 1 2"

Article Title: Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes 1Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes 1 2

Journal: Neoplasia (New York, N.Y.)

doi:

Construction and expression of the EGFR-specific CAR in transduced human T lymphocytes. (A and B) Schematic representation of the construction of EGFR-specific CAR. This CAR consisted of the hIL-2 signal peptide, anti-EGFR scFv, HA tag, IgG2 Fc, the transmembrane region, and part of the cytoplasmic region of the human CD28 signaling chain, fused to the cytoplasmic region of the CD3ζ chain. This CAR gene was cloned into Nhe I and Bgl II sites of a CMV promoter-based nonviral vector, pmaxCloning. (C) Western blot analysis of CAR expression in T lymphocytes at 16 hours after transduction. PVDF membranes were labeled with an anti-human CD3ζ antibody and a goat anti-rabbit IgG HRP-conjugated second antibody. (D) Flow cytometric analysis of CAR expression in T lymphocytes at 16 hours after transduction. The transduced cells were stained with anti-EGFR scFv antiserum and goat anti-rabbit IgG FITC-conjugated secondary antibody and were then detected by a BD FACSCalibur flow cytometer. MFI is the abbreviation of mean fluorescence intensity. Results are representative of three experiments.
Figure Legend Snippet: Construction and expression of the EGFR-specific CAR in transduced human T lymphocytes. (A and B) Schematic representation of the construction of EGFR-specific CAR. This CAR consisted of the hIL-2 signal peptide, anti-EGFR scFv, HA tag, IgG2 Fc, the transmembrane region, and part of the cytoplasmic region of the human CD28 signaling chain, fused to the cytoplasmic region of the CD3ζ chain. This CAR gene was cloned into Nhe I and Bgl II sites of a CMV promoter-based nonviral vector, pmaxCloning. (C) Western blot analysis of CAR expression in T lymphocytes at 16 hours after transduction. PVDF membranes were labeled with an anti-human CD3ζ antibody and a goat anti-rabbit IgG HRP-conjugated second antibody. (D) Flow cytometric analysis of CAR expression in T lymphocytes at 16 hours after transduction. The transduced cells were stained with anti-EGFR scFv antiserum and goat anti-rabbit IgG FITC-conjugated secondary antibody and were then detected by a BD FACSCalibur flow cytometer. MFI is the abbreviation of mean fluorescence intensity. Results are representative of three experiments.

Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Western Blot, Transduction, Labeling, Flow Cytometry, Staining, Cytometry, Fluorescence

28) Product Images from "Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5"

Article Title: Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5

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

doi: 10.1073/pnas.0809485106

Tamoxifen-dependent activation of mb-1 gene expression. ( A ) Pax5:ER increases the display of mIgM on μM.10 cells. Cells were infected with Pax5:ER-YFP retroviruses. YFP + cells were sorted and 0.5 μM 4-OHT was added at 48 h posttransduction. 48 h after the addition of 4-OHT, cell surface expression of mIgM (which directly reflects mb-1 expression) was analyzed by labeling cells with biotinylated anti-IgM and streptavidin-conjugated APC. mIgM was detected using a FACScalibur™ flow cytometer. ( B ) Synergistic activation of mb-1 transcription by EBF:ER and Pax5 in μM.2 cells. Cells were stably transfected to express EBF:ER (μM.2+EBF:ER cells), then infected with MSCV-Pax5-GFP retroviruses. Cells are shown following incubation with or without 4-OHT and mIgM staining as in A .
Figure Legend Snippet: Tamoxifen-dependent activation of mb-1 gene expression. ( A ) Pax5:ER increases the display of mIgM on μM.10 cells. Cells were infected with Pax5:ER-YFP retroviruses. YFP + cells were sorted and 0.5 μM 4-OHT was added at 48 h posttransduction. 48 h after the addition of 4-OHT, cell surface expression of mIgM (which directly reflects mb-1 expression) was analyzed by labeling cells with biotinylated anti-IgM and streptavidin-conjugated APC. mIgM was detected using a FACScalibur™ flow cytometer. ( B ) Synergistic activation of mb-1 transcription by EBF:ER and Pax5 in μM.2 cells. Cells were stably transfected to express EBF:ER (μM.2+EBF:ER cells), then infected with MSCV-Pax5-GFP retroviruses. Cells are shown following incubation with or without 4-OHT and mIgM staining as in A .

Techniques Used: Activation Assay, Expressing, Infection, Labeling, Flow Cytometry, Cytometry, Stable Transfection, Transfection, Incubation, Staining

29) Product Images from "Glioblastoma entities express subtle differences in molecular composition and response to treatment"

Article Title: Glioblastoma entities express subtle differences in molecular composition and response to treatment

Journal: Oncology Reports

doi: 10.3892/or.2017.5799

Effects of TMX and TMZ combination on EdU incorporation and p-PKC expression of GBM11 cells. (A) The proliferation rate was evaluated by measuring the incorporation of EdU. Analysis were performed with a BD FACSCalibur system evaluated in the FL2 and FL1 channel and a total of 10,000 events were collected. (B) The effect of TMX and/or TMZ on PKC-pan expression was evaluated by western blot analysis. Loading control was performed with an antibody for α-tubulin. The bands numbered from 1 to 6 were in the same order that they appear in the graph. Each value represents the mean ± SEM from three independent experiments, *P
Figure Legend Snippet: Effects of TMX and TMZ combination on EdU incorporation and p-PKC expression of GBM11 cells. (A) The proliferation rate was evaluated by measuring the incorporation of EdU. Analysis were performed with a BD FACSCalibur system evaluated in the FL2 and FL1 channel and a total of 10,000 events were collected. (B) The effect of TMX and/or TMZ on PKC-pan expression was evaluated by western blot analysis. Loading control was performed with an antibody for α-tubulin. The bands numbered from 1 to 6 were in the same order that they appear in the graph. Each value represents the mean ± SEM from three independent experiments, *P

Techniques Used: Expressing, Western Blot

Effects of TMX and TMZ combination on cell death and cell cycle of GBM11 cells. Following the incubation of GBM11 cells with TMX and/or TMZ for 48 h, cells were stained with Annexin V (AV) and propidium iodide (PI) and analysed by flow cytometry. (A) Debris was removed to obtain the viable cell region, R1. The R1 region was used to analyse AV and PI expression by flow cytometry. The analysis was based on the percentage of gated positive cells. (B) TMZ alone was added to GBM11 cells in different concentrations. (C) TMX and/or in combination with TMZ was added to GBM11 cells in different concentrations. The chosen doses were above the dose that inhibited growth by 50% (IC 50 ), respectively. The AV positive cells, PI positive cells, AV and PI double-positive cells and the live cells (double-negative) were immediately analysed by flow cytometry in a BD FACSCalibur system and evaluated in the FL2 and FL1 channel, respectively. A total of 10,000 events were collected. (D) Cell cycle analysis was determined by gating G0/G1, S and G2/M on PI-area signal by flow cytometry after the debris were removed to obtain the R1 region. *P
Figure Legend Snippet: Effects of TMX and TMZ combination on cell death and cell cycle of GBM11 cells. Following the incubation of GBM11 cells with TMX and/or TMZ for 48 h, cells were stained with Annexin V (AV) and propidium iodide (PI) and analysed by flow cytometry. (A) Debris was removed to obtain the viable cell region, R1. The R1 region was used to analyse AV and PI expression by flow cytometry. The analysis was based on the percentage of gated positive cells. (B) TMZ alone was added to GBM11 cells in different concentrations. (C) TMX and/or in combination with TMZ was added to GBM11 cells in different concentrations. The chosen doses were above the dose that inhibited growth by 50% (IC 50 ), respectively. The AV positive cells, PI positive cells, AV and PI double-positive cells and the live cells (double-negative) were immediately analysed by flow cytometry in a BD FACSCalibur system and evaluated in the FL2 and FL1 channel, respectively. A total of 10,000 events were collected. (D) Cell cycle analysis was determined by gating G0/G1, S and G2/M on PI-area signal by flow cytometry after the debris were removed to obtain the R1 region. *P

Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Expressing, Cell Cycle Assay

Evaluation of the resistance mechanisms in GBM cell lines. (A) Nestin, GFAP and phalloidin positive-staining were analysed for U87 and U118 cell lines. (B) PGP expression was quantified in the three GBM cell lines, U87, U118 and GBM11 by flow cytometric analysis in a BD FACSCalibur system. A total of 10,000 events were collected. Relative expression was obtained by PGP expression compared to the respective isotype and corresponds to the % of gated cells. (C) MGMT methylation pattern was analysed through methylation-specific PCRs for U87, U118 and GBM11 cells lines. UC, universal unmethylated control (bisulfite converted); MC, universal methylated control (bisulfite converted); UNC, universal not converted unmethylated control (not bisulfite converted); MM, molecular marker of 50 bp. (D) The expression of SOX2, Oct-4A and Nanog in U87, U118 and GBM11 cell lines was quantified by western blot analysis. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA, USA). Each value represents the mean ± SEM from three independent experiments, *P
Figure Legend Snippet: Evaluation of the resistance mechanisms in GBM cell lines. (A) Nestin, GFAP and phalloidin positive-staining were analysed for U87 and U118 cell lines. (B) PGP expression was quantified in the three GBM cell lines, U87, U118 and GBM11 by flow cytometric analysis in a BD FACSCalibur system. A total of 10,000 events were collected. Relative expression was obtained by PGP expression compared to the respective isotype and corresponds to the % of gated cells. (C) MGMT methylation pattern was analysed through methylation-specific PCRs for U87, U118 and GBM11 cells lines. UC, universal unmethylated control (bisulfite converted); MC, universal methylated control (bisulfite converted); UNC, universal not converted unmethylated control (not bisulfite converted); MM, molecular marker of 50 bp. (D) The expression of SOX2, Oct-4A and Nanog in U87, U118 and GBM11 cell lines was quantified by western blot analysis. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA, USA). Each value represents the mean ± SEM from three independent experiments, *P

Techniques Used: Staining, Expressing, Flow Cytometry, Methylation, Marker, Western Blot, Software

30) Product Images from "Calcium-independent Phospholipase A2 Localizes in and Protects Mitochondria during Apoptotic Induction by Staurosporine *"

Article Title: Calcium-independent Phospholipase A2 Localizes in and Protects Mitochondria during Apoptotic Induction by Staurosporine *

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M604330200

STS induces apoptosis, mitochondrial ROS generation, and membrane lipid peroxidation in INS-1 cells . A , FACS analysis of apoptosis as indicated by PS externalization. INS-1 cells were treated with or without 1 μM STS for 8 h. Cells were collected and stained with an annexin V ( AnnV )-FLUOS labeling solution containing annexin V and PI, analyzed by FACS ( left panel ), and results are summarized ( right panel ). R1 represents annexin V-positive and PI-negative cells, and R2 represents annexin V- and PI-positive cells. B , mitochondrial ROS production. Cells were collected and stained with 2 μM HE and analyzed on a BD Biosciences FACSCalibur. Representative histograms obtained are presented. The left histogram shows autofluorescence, and the right histogram shows cells containing converted HE. C , membrane lipid peroxidation. Cells were collected and treated with trichloroacetic acid/TBARS reagent. Each sample was incubated at 95 °C for 60 min. The supernatants were analyzed, and values are expressed as a percentage of control values. Data are the averages ± S.D. ( n = 4). * , p
Figure Legend Snippet: STS induces apoptosis, mitochondrial ROS generation, and membrane lipid peroxidation in INS-1 cells . A , FACS analysis of apoptosis as indicated by PS externalization. INS-1 cells were treated with or without 1 μM STS for 8 h. Cells were collected and stained with an annexin V ( AnnV )-FLUOS labeling solution containing annexin V and PI, analyzed by FACS ( left panel ), and results are summarized ( right panel ). R1 represents annexin V-positive and PI-negative cells, and R2 represents annexin V- and PI-positive cells. B , mitochondrial ROS production. Cells were collected and stained with 2 μM HE and analyzed on a BD Biosciences FACSCalibur. Representative histograms obtained are presented. The left histogram shows autofluorescence, and the right histogram shows cells containing converted HE. C , membrane lipid peroxidation. Cells were collected and treated with trichloroacetic acid/TBARS reagent. Each sample was incubated at 95 °C for 60 min. The supernatants were analyzed, and values are expressed as a percentage of control values. Data are the averages ± S.D. ( n = 4). * , p

Techniques Used: FACS, Staining, Labeling, Incubation

31) Product Images from "2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation, et al. 2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation"

Article Title: 2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation, et al. 2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation

Journal: Cancer Science

doi: 10.1111/cas.13852

2′‐Hydroxycinnamaldehyde (HCA) induces reactive oxygen species (ROS) generation by regulating ERK1/2 and signal transducer and activator of transcription 3 (STAT3) activation. A, DU145 cells were treated with HCA (20 μmol/L) for the indicated times. After treatment, intracellular ROS levels were measured by DCF‐DA (5 μmol/L) using a FACSCalibur flow cytometer. B, DU145 cells were treated with HCA (20 μmol/L) for the indicated times, and the expression level of p‐ERK1/2 (T202/Y204) and ERK1/2 was analyzed by western blotting (n = 2). C,D, After pre‐treatment with N‐acetyl‐L‐cysteine (NAC) (2 mmol/L) or glutathione (GSH) (2 mmol/L) for 1 h, DU145 cells were treated with HCA for 1 h (C) or 24 h (D) in the presence or absence of NAC or GSH. The expression level of p‐STAT3 (Y705), STAT3, p‐ERK1/2 (T202/Y204) and ERK1/2 was analyzed by western blotting (n = 2) (C). Cell proliferation was measured by WST‐1 (n = 3) (D). E, CETSA in DU145 cells that were treated with HCA (20 μmol/L) for 1 h in the presence or absence of NAC (2 mmol/L) or GSH (2 mmol/L) at a temperature of 58 or 37°C (n = 2). The band intensity of each protein was quantified using the MultiGauge program. ** P
Figure Legend Snippet: 2′‐Hydroxycinnamaldehyde (HCA) induces reactive oxygen species (ROS) generation by regulating ERK1/2 and signal transducer and activator of transcription 3 (STAT3) activation. A, DU145 cells were treated with HCA (20 μmol/L) for the indicated times. After treatment, intracellular ROS levels were measured by DCF‐DA (5 μmol/L) using a FACSCalibur flow cytometer. B, DU145 cells were treated with HCA (20 μmol/L) for the indicated times, and the expression level of p‐ERK1/2 (T202/Y204) and ERK1/2 was analyzed by western blotting (n = 2). C,D, After pre‐treatment with N‐acetyl‐L‐cysteine (NAC) (2 mmol/L) or glutathione (GSH) (2 mmol/L) for 1 h, DU145 cells were treated with HCA for 1 h (C) or 24 h (D) in the presence or absence of NAC or GSH. The expression level of p‐STAT3 (Y705), STAT3, p‐ERK1/2 (T202/Y204) and ERK1/2 was analyzed by western blotting (n = 2) (C). Cell proliferation was measured by WST‐1 (n = 3) (D). E, CETSA in DU145 cells that were treated with HCA (20 μmol/L) for 1 h in the presence or absence of NAC (2 mmol/L) or GSH (2 mmol/L) at a temperature of 58 or 37°C (n = 2). The band intensity of each protein was quantified using the MultiGauge program. ** P

Techniques Used: High Content Screening, Activation Assay, Flow Cytometry, Cytometry, Expressing, Western Blot

2′‐Hydroxycinnamaldehyde (HCA) downregulates the expression of signal transducer and activator of transcription 3 (STAT3) target genes that induces G0/G1 arrest and apoptosis. A, DU145 cells were treated with HCA (20 μmol/L) for 24 or 48 h and the expression level of the STAT3 target gene was analyzed by western blotting (n = 2). The band intensity of each protein was quantified using the MultiGauge program. B, DU145 cells were treated with HCA (20 μmol/L) for the indicated times. After treatment, the cell cycle distribution was analyzed using a FACSCalibur flow cytometer (n = 2). The ratios of cells in each phase were analyzed using the WinMDI 2.9 analyzer
Figure Legend Snippet: 2′‐Hydroxycinnamaldehyde (HCA) downregulates the expression of signal transducer and activator of transcription 3 (STAT3) target genes that induces G0/G1 arrest and apoptosis. A, DU145 cells were treated with HCA (20 μmol/L) for 24 or 48 h and the expression level of the STAT3 target gene was analyzed by western blotting (n = 2). The band intensity of each protein was quantified using the MultiGauge program. B, DU145 cells were treated with HCA (20 μmol/L) for the indicated times. After treatment, the cell cycle distribution was analyzed using a FACSCalibur flow cytometer (n = 2). The ratios of cells in each phase were analyzed using the WinMDI 2.9 analyzer

Techniques Used: High Content Screening, Expressing, Western Blot, Flow Cytometry, Cytometry

32) Product Images from "Performance Evaluation of the MBio Diagnostics Point-of-Care CD4 Counter"

Article Title: Performance Evaluation of the MBio Diagnostics Point-of-Care CD4 Counter

Journal: Journal of immunological methods

doi: 10.1016/j.jim.2012.10.002

SnapCount™ performance with venous blood samples (A) Scatter plot of SnapCount ™ versus FACSCalibur CD4 cell counts. The solid line is the identity line. (B) Bland-Altman plot comparing SnapCount ™ and FACSCalibur. (C) Bland-Altman parameters. (D) Histogram for percentage similarity (%SIM) of SnapCount ™ and FACSCalibur. The solid curve is a normal distribution fitted to the histogram, which yields a %SIM of 99.8% ± 7.3% and a 7.3%SIM CV.
Figure Legend Snippet: SnapCount™ performance with venous blood samples (A) Scatter plot of SnapCount ™ versus FACSCalibur CD4 cell counts. The solid line is the identity line. (B) Bland-Altman plot comparing SnapCount ™ and FACSCalibur. (C) Bland-Altman parameters. (D) Histogram for percentage similarity (%SIM) of SnapCount ™ and FACSCalibur. The solid curve is a normal distribution fitted to the histogram, which yields a %SIM of 99.8% ± 7.3% and a 7.3%SIM CV.

Techniques Used:

SnapCount™ performance with capillary blood samples (A) Scatter plot of SnapCount ™ versus FACSCalibur CD4 cell counts. The identity line is indicated with the solid line. (B) Bland-Altman plot comparing SnapCount ™ and FACSCalibur. (C) Bland-Altman parameters. (D) Histogram for percentage similarity (%SIM) to FACSCalibur CD4 cell counts. One sample with CD4 count
Figure Legend Snippet: SnapCount™ performance with capillary blood samples (A) Scatter plot of SnapCount ™ versus FACSCalibur CD4 cell counts. The identity line is indicated with the solid line. (B) Bland-Altman plot comparing SnapCount ™ and FACSCalibur. (C) Bland-Altman parameters. (D) Histogram for percentage similarity (%SIM) to FACSCalibur CD4 cell counts. One sample with CD4 count

Techniques Used:

33) Product Images from "Loss of Hypoxia-Inducible Factor 2 Alpha in the Lung Alveolar Epithelium of Mice Leads to Enhanced Eosinophilic Inflammation in Cobalt-Induced Lung Injury"

Article Title: Loss of Hypoxia-Inducible Factor 2 Alpha in the Lung Alveolar Epithelium of Mice Leads to Enhanced Eosinophilic Inflammation in Cobalt-Induced Lung Injury

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kft253

LL cytokine analysis. LL was analyzed for cytokine protein levels using cytokine bead array and FACSCalibur flow cytometer (BD Biosciences). Shown are IL-4 (A), IL-5 (B), IL-10 (C), KC (D), tumor necrosis factor alpha (E), and IFNγ (F). Open bars
Figure Legend Snippet: LL cytokine analysis. LL was analyzed for cytokine protein levels using cytokine bead array and FACSCalibur flow cytometer (BD Biosciences). Shown are IL-4 (A), IL-5 (B), IL-10 (C), KC (D), tumor necrosis factor alpha (E), and IFNγ (F). Open bars

Techniques Used: Flow Cytometry, Cytometry

34) Product Images from "CXCR7 participates in CXCL12-mediated migration and homing of leukemic and normal hematopoietic cells"

Article Title: CXCR7 participates in CXCL12-mediated migration and homing of leukemic and normal hematopoietic cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0765-1

CXCR7 participates in U937 and normal CD34 + cell migration and prevents downregulation of CXCR4 by CXCL12 stimulation. CXCR4 and CXCR7 extracellular and intracellular expression was analyzed in CD34 ( a , b ) and U937 ( c , d ) cells on a FACScalibur flow cytometer after labeling with specific antibodies. Results are representative of one experiment of the three to five performed. Histograms show the percentage of cells expressing CXCR4 and CXCR7. The U937 cell line was chosen due to the CXCR4 and CXCR7 expression and high migration capability demonstrated towards CXCL12 ( e ). Transwell assay shows cell migration toward RPMI + 0.5% BSA, containing or not CXCL12 (200 ng/mL). The number of migrated cells (16 h for U937 and 6 h for CD34 + ) is expressed as a percentage of the input. The migration of cells was normalized to 100% ± the standard deviation of triplicates. f There was a significant reduction in sh CXCR7 U937 cell migration compared to shControl U937 cells. Blocking of CXCR4 by CXCR4 mAb-clone 12G5 promoted a similar effect. Moreover, silencing of CXCR7 plus CXCR4 mAb treatment inhibited cell chemotactic capacity. g The same effect was observed for normal CD34 + cells, i.e., blockage of CXCR7 by CXCR7 mAb-clone 11G8 or CXCR4 or both receptors together also promoted a reduction in cell migration. h shControl and sh CXCR7 U937 cells were stimulated with CXCL12, treated or not with phosphatase, and then labeled with anti-CXCR4 UMB-2. This antibody recognizes non-phosphorylated C-terminus. Thus, dephosphorylated samples (using phosphatase) show total CXCR4, whereas untreated aliquots show inactive CXCR4. This figure shows that, in shControl U937 cells, induction with CXCL12 caused activation of CXCR4, since the inactive form does not appear or is very low, which is characteristic of CXCR4 activation ( column A ). On the other hand, sh CXCR7 U937 cells induced by CXCL12 showed no or low expression of total CXCR4 ( column D ), suggesting that CXCR7 is important for preventing downregulation of CXCR4 in leukemia cells. Anti-transferrin receptor ( TfR ) was used as control to ensure equal loading. Data represent three independent experiments.* p
Figure Legend Snippet: CXCR7 participates in U937 and normal CD34 + cell migration and prevents downregulation of CXCR4 by CXCL12 stimulation. CXCR4 and CXCR7 extracellular and intracellular expression was analyzed in CD34 ( a , b ) and U937 ( c , d ) cells on a FACScalibur flow cytometer after labeling with specific antibodies. Results are representative of one experiment of the three to five performed. Histograms show the percentage of cells expressing CXCR4 and CXCR7. The U937 cell line was chosen due to the CXCR4 and CXCR7 expression and high migration capability demonstrated towards CXCL12 ( e ). Transwell assay shows cell migration toward RPMI + 0.5% BSA, containing or not CXCL12 (200 ng/mL). The number of migrated cells (16 h for U937 and 6 h for CD34 + ) is expressed as a percentage of the input. The migration of cells was normalized to 100% ± the standard deviation of triplicates. f There was a significant reduction in sh CXCR7 U937 cell migration compared to shControl U937 cells. Blocking of CXCR4 by CXCR4 mAb-clone 12G5 promoted a similar effect. Moreover, silencing of CXCR7 plus CXCR4 mAb treatment inhibited cell chemotactic capacity. g The same effect was observed for normal CD34 + cells, i.e., blockage of CXCR7 by CXCR7 mAb-clone 11G8 or CXCR4 or both receptors together also promoted a reduction in cell migration. h shControl and sh CXCR7 U937 cells were stimulated with CXCL12, treated or not with phosphatase, and then labeled with anti-CXCR4 UMB-2. This antibody recognizes non-phosphorylated C-terminus. Thus, dephosphorylated samples (using phosphatase) show total CXCR4, whereas untreated aliquots show inactive CXCR4. This figure shows that, in shControl U937 cells, induction with CXCL12 caused activation of CXCR4, since the inactive form does not appear or is very low, which is characteristic of CXCR4 activation ( column A ). On the other hand, sh CXCR7 U937 cells induced by CXCL12 showed no or low expression of total CXCR4 ( column D ), suggesting that CXCR7 is important for preventing downregulation of CXCR4 in leukemia cells. Anti-transferrin receptor ( TfR ) was used as control to ensure equal loading. Data represent three independent experiments.* p

Techniques Used: Migration, Expressing, Flow Cytometry, Cytometry, Labeling, Transwell Assay, Standard Deviation, Blocking Assay, Activation Assay

35) Product Images from "HS-173, a Novel PI3K Inhibitor, Attenuates the Activation of Hepatic Stellate Cells in Liver Fibrosis"

Article Title: HS-173, a Novel PI3K Inhibitor, Attenuates the Activation of Hepatic Stellate Cells in Liver Fibrosis

Journal: Scientific Reports

doi: 10.1038/srep03470

Effect of HS-173 on the HSC cell cycle. (A) After incubating for 1 day, HSC-T6 cells were treated with various concentrations of HS-173 (0, 0.1, 1, and 5 μM) for 8 h, stained with propidium iodide (PI) and analyzed with a FACSCalibur flow cytometer. M1, sub-G1; M2, G0/G1; M3, S; and M4, G2/M. Quantitation of the PI staining data is presented as the cell cycle distribution percentages. (B) The expression of p-cdc2 and cyclin B1 was evaluated by immunofluorescence in HSC-T6 cells treated with 5 μM of HS-173 for 8 h. 400 × and 800 × magnification.
Figure Legend Snippet: Effect of HS-173 on the HSC cell cycle. (A) After incubating for 1 day, HSC-T6 cells were treated with various concentrations of HS-173 (0, 0.1, 1, and 5 μM) for 8 h, stained with propidium iodide (PI) and analyzed with a FACSCalibur flow cytometer. M1, sub-G1; M2, G0/G1; M3, S; and M4, G2/M. Quantitation of the PI staining data is presented as the cell cycle distribution percentages. (B) The expression of p-cdc2 and cyclin B1 was evaluated by immunofluorescence in HSC-T6 cells treated with 5 μM of HS-173 for 8 h. 400 × and 800 × magnification.

Techniques Used: Staining, Flow Cytometry, Cytometry, Quantitation Assay, Expressing, Immunofluorescence

36) Product Images from "Combining α-Radioimmunotherapy and Adoptive T Cell Therapy to Potentiate Tumor Destruction"

Article Title: Combining α-Radioimmunotherapy and Adoptive T Cell Therapy to Potentiate Tumor Destruction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0130249

5T33-OVA phenotypic analysis after transduction by a lentiviral vector encoding cytoplasmic ovalbumin (A) 5T33 and 5T33-OVA staining with PE-conjugated antibody 25-D1.16, which specifically recognizes the OVA peptide ‘SIINFEKL’ bound to the MHC class I molecule H-2K b and (B) staining of 5T33 and 5T33-OVA with APC-conjugated anti-mouse CD138 mAb. Flow cytometry was performed on a BD FACSCalibur Flow Cytometry System.
Figure Legend Snippet: 5T33-OVA phenotypic analysis after transduction by a lentiviral vector encoding cytoplasmic ovalbumin (A) 5T33 and 5T33-OVA staining with PE-conjugated antibody 25-D1.16, which specifically recognizes the OVA peptide ‘SIINFEKL’ bound to the MHC class I molecule H-2K b and (B) staining of 5T33 and 5T33-OVA with APC-conjugated anti-mouse CD138 mAb. Flow cytometry was performed on a BD FACSCalibur Flow Cytometry System.

Techniques Used: Transduction, Plasmid Preparation, Staining, Flow Cytometry, Cytometry

37) Product Images from "The long noncoding RNA GAS8-AS1 suppresses hepatocarcinogenesis by epigenetically activating the tumor suppressor GAS8"

Article Title: The long noncoding RNA GAS8-AS1 suppresses hepatocarcinogenesis by epigenetically activating the tumor suppressor GAS8

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.003055

lncRNA GAS8-AS1 and GAS8 induce apoptosis of HepG2 or SMMC7721 cells via sensitizing them to sorafenib. HepG2 or SMMC7721 cells were transfected with expression constructs or siRNAs of GAS8-AS1 or GAS8 first. At the 24th hour after transfection, HCC cells were treated with 1 μmol/liter sorafenib, and apoptosis was determined with a FACSCalibur flow cytometer. All results of the mean of triplicate assays with S.D. ( error bars ) are presented. *, p
Figure Legend Snippet: lncRNA GAS8-AS1 and GAS8 induce apoptosis of HepG2 or SMMC7721 cells via sensitizing them to sorafenib. HepG2 or SMMC7721 cells were transfected with expression constructs or siRNAs of GAS8-AS1 or GAS8 first. At the 24th hour after transfection, HCC cells were treated with 1 μmol/liter sorafenib, and apoptosis was determined with a FACSCalibur flow cytometer. All results of the mean of triplicate assays with S.D. ( error bars ) are presented. *, p

Techniques Used: Transfection, Expressing, Construct, Flow Cytometry, Cytometry

38) Product Images from "Up-regulation of CNDP2 facilitates the proliferation of colon cancer"

Article Title: Up-regulation of CNDP2 facilitates the proliferation of colon cancer

Journal: BMC Gastroenterology

doi: 10.1186/1471-230X-14-96

Association of CN2 expression with colon cancer cell cycle progression and tumorigenicity. (A) Cell-cycle phase distributions were analyzed by a FACScalibur flow cytometer. These experiments were repeated 3 times, and the symbols represent the mean values of triplicate tests (mean ± SD). (B) Western blotting analysis of cyclin E, cyclin B1 and EGFR expression. The experiment was independently repeated at least 3 times. (C) Knockdown of CNDP2 inhibited tumor formation in nude mice. Nude mice were inoculated with RKO stably transfected with empty vector and shRNA_CNDP2 expression vectors. *p
Figure Legend Snippet: Association of CN2 expression with colon cancer cell cycle progression and tumorigenicity. (A) Cell-cycle phase distributions were analyzed by a FACScalibur flow cytometer. These experiments were repeated 3 times, and the symbols represent the mean values of triplicate tests (mean ± SD). (B) Western blotting analysis of cyclin E, cyclin B1 and EGFR expression. The experiment was independently repeated at least 3 times. (C) Knockdown of CNDP2 inhibited tumor formation in nude mice. Nude mice were inoculated with RKO stably transfected with empty vector and shRNA_CNDP2 expression vectors. *p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Western Blot, Mouse Assay, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Association of CN2 expression with cell apoptosis. Cell apoptosis was analyzed by a FACScalibur flow cytometer. These experiments were repeated 3 times, and the symbols represent the mean values of triplicate tests (mean ± SD).
Figure Legend Snippet: Association of CN2 expression with cell apoptosis. Cell apoptosis was analyzed by a FACScalibur flow cytometer. These experiments were repeated 3 times, and the symbols represent the mean values of triplicate tests (mean ± SD).

Techniques Used: Expressing, Flow Cytometry, Cytometry

39) Product Images from "Attenuation of niacin-induced prostaglandin D2 generation by omega-3 fatty acids in THP-1 macrophages and Langerhans dendritic cells"

Article Title: Attenuation of niacin-induced prostaglandin D2 generation by omega-3 fatty acids in THP-1 macrophages and Langerhans dendritic cells

Journal: Journal of Inflammation Research

doi: 10.2147/JIR.S29044

Effect of fatty acids on GPR109A niacin receptor expression in THP-1 macrophages. Notes: Macrophages were treated with 50 or 75 μM DHA, EPA, and OLA for 24 hours prior to stimulation with niacin (30 minutes). Cells were labeled with phycoerythrin-conjugated GPR109A and expression of GPR109A was determined using a FACSCalibur flow cytometer as described in the text. ( A ) Filled histograms represent the isotype control and open histograms represent GPR109A. Data is representative of the 75 μM treatments. ( B ) Data were quantified as the percent change of mean fluorescent intensity. Values are the means ± the standard deviations of three independent duplicate experiments. Results are analyzed using ANOVA, followed by pair-wise comparisons with the Bonferroni adjustment. * P
Figure Legend Snippet: Effect of fatty acids on GPR109A niacin receptor expression in THP-1 macrophages. Notes: Macrophages were treated with 50 or 75 μM DHA, EPA, and OLA for 24 hours prior to stimulation with niacin (30 minutes). Cells were labeled with phycoerythrin-conjugated GPR109A and expression of GPR109A was determined using a FACSCalibur flow cytometer as described in the text. ( A ) Filled histograms represent the isotype control and open histograms represent GPR109A. Data is representative of the 75 μM treatments. ( B ) Data were quantified as the percent change of mean fluorescent intensity. Values are the means ± the standard deviations of three independent duplicate experiments. Results are analyzed using ANOVA, followed by pair-wise comparisons with the Bonferroni adjustment. * P

Techniques Used: Expressing, Labeling, Flow Cytometry, Cytometry

40) Product Images from "Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III"

Article Title: Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00059

Antibodies from mice immunized with pscDEC-EDIII and pscISO-EDIII partially inhibit DENV infection by binding to EDIII. (A) Neutralization in VERO cells. Pooled sera from mice immunized as described in Figure 2 were heat inactivated at 56°C for 30 min. Two-fold serially diluted sera were incubated with the DENV2 particles for 30 min at 37°C and 5% CO 2 . The sera/virus mixture was then incubated with the cells for 1 h at 37°C and 5% CO 2 . The cultures supernatant was replaced by DMEM 5% FBS followed by another incubation at the same conditions for 24 h. Cells were stained with the 4G2 (mouse anti-flavivirus envelope antibody) and anti-mouse IgG-Alexa488. The neutralization of infection was determined in comparison to a control infection. Results are represented by means and SEM from pooled data of four independent experiments. (B) Competition assay with the recombinant EDIII protein. A fixed dilution (1:20) of sera was incubated with increasing molar concentrations of EDIII protein prior to incubation with the virus. The cells were resuspended in FACS buffer and 20,000 events were acquired in the BD FACSCalibur™ (BD biosciences) flow cytometer. The neutralization of infection was determined in comparison to a control infection with sera derived from mice injected with saline. Data were analyzed by a two-way ANOVA for repetitive measures followed by Sidak's multiple comparisons test. ns = not-significant. Representative of three independent experiments.
Figure Legend Snippet: Antibodies from mice immunized with pscDEC-EDIII and pscISO-EDIII partially inhibit DENV infection by binding to EDIII. (A) Neutralization in VERO cells. Pooled sera from mice immunized as described in Figure 2 were heat inactivated at 56°C for 30 min. Two-fold serially diluted sera were incubated with the DENV2 particles for 30 min at 37°C and 5% CO 2 . The sera/virus mixture was then incubated with the cells for 1 h at 37°C and 5% CO 2 . The cultures supernatant was replaced by DMEM 5% FBS followed by another incubation at the same conditions for 24 h. Cells were stained with the 4G2 (mouse anti-flavivirus envelope antibody) and anti-mouse IgG-Alexa488. The neutralization of infection was determined in comparison to a control infection. Results are represented by means and SEM from pooled data of four independent experiments. (B) Competition assay with the recombinant EDIII protein. A fixed dilution (1:20) of sera was incubated with increasing molar concentrations of EDIII protein prior to incubation with the virus. The cells were resuspended in FACS buffer and 20,000 events were acquired in the BD FACSCalibur™ (BD biosciences) flow cytometer. The neutralization of infection was determined in comparison to a control infection with sera derived from mice injected with saline. Data were analyzed by a two-way ANOVA for repetitive measures followed by Sidak's multiple comparisons test. ns = not-significant. Representative of three independent experiments.

Techniques Used: Mouse Assay, Infection, Binding Assay, Neutralization, Incubation, Staining, Competitive Binding Assay, Recombinant, FACS, Flow Cytometry, Cytometry, Derivative Assay, Injection

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Flow Cytometry:

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Article Title: Heparan sulfate structure is influenced by the ER-Golgi dynamics of its modifying enzymes
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Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection
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Cytometry:

Article Title: Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region
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Blocking Assay:

Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection
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Incubation:

Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection
Article Snippet: .. Cells were incubated with anti-CD16/CD32 Fc block (BD PharMingen) for 10 min on ice; staining with tetrameric reagents took place for 1 h at room temperature, followed by staining with anti-CD8a PerCP (clone 53-6.7) and anti-CD62L FITC (clone MEL-14) or anti-CD43 PE (clone 1B11) on ice for 20 min. Stained samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Immunocytometry Systems, Mountain View, CA). .. Control tetramers consisting of the same H chain folded with irrelevant peptides did not stain CD8 T cells from MHV-68 infected mice.

Staining:

Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection
Article Snippet: .. Cells were incubated with anti-CD16/CD32 Fc block (BD PharMingen) for 10 min on ice; staining with tetrameric reagents took place for 1 h at room temperature, followed by staining with anti-CD8a PerCP (clone 53-6.7) and anti-CD62L FITC (clone MEL-14) or anti-CD43 PE (clone 1B11) on ice for 20 min. Stained samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Immunocytometry Systems, Mountain View, CA). .. Control tetramers consisting of the same H chain folded with irrelevant peptides did not stain CD8 T cells from MHV-68 infected mice.

Software:

Article Title: Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region
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Article Title: Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms
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Article Title: Major histocompatibility complex class I molecules with super-enhanced CD8 binding properties bypass the requirement for cognate TCR recognition and non-specifically activate cytotoxic T lymphocytes 1
Article Snippet: .. Data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest software (BD BioSciences). .. CTL were incubated with C1R A2 cells, C1R A2/Kb cells or medium alone at different effector:target (E:T) ratios overnight at 37°C.

Article Title: Heparan sulfate structure is influenced by the ER-Golgi dynamics of its modifying enzymes
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Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection
Article Snippet: .. Cells were incubated with anti-CD16/CD32 Fc block (BD PharMingen) for 10 min on ice; staining with tetrameric reagents took place for 1 h at room temperature, followed by staining with anti-CD8a PerCP (clone 53-6.7) and anti-CD62L FITC (clone MEL-14) or anti-CD43 PE (clone 1B11) on ice for 20 min. Stained samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Immunocytometry Systems, Mountain View, CA). .. Control tetramers consisting of the same H chain folded with irrelevant peptides did not stain CD8 T cells from MHV-68 infected mice.

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    Becton Dickinson facscalibur flow cytometer
    Vanillic acid inhibits the proliferation of HCT116 cells via blocking cell cycle progression in the G1 phase. ( A ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Subsequently, cell cycle status was analyzed by by <t>FACSCalibur</t> flow cytometry. ( B ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Cyclin D1 and c-Myc were detected by Western blot analysis. Levels of tubulin were used as a loading control. ( C ) Data were shown as mean ± SD ( n = 3). ** p
    Facscalibur Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 18987 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oral tolerance induction in indoleamine 2,3-dioxygenase-expressing CD11c + dendritic cells of tolerized mice. The induction of oral tolerance increases the proportion of indoleamine 2,3-dioxygenase (IDO)-expressing CD11c + dendritic cells (DCs) in Peyer's patches of tolerized mice. (a) Flow <t>cytometric</t> analysis of IDO in CD11c + DCs isolated from Peyer's patches. Mononuclear cells obtained from Peyer's patches of tolerized mice and of CIA mice were probed with Fluorescein isothiocyanate-labeled anti-CD11c mAb and were fixed with CytoPerm/CytoFix for 20 minutes. Cells were probed for intracellular IDO using anti-mouse IDO antibody and were analyzed by flow cytometry. The histograms were gated on CD11c + DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Results are the mean ± standard deviation of replicate samples from seven independent experiments. SSC. (b) Analysis of IDO transcription in tolerized mice and CIA mice. CD11c + DCs were isolated from Peyer's patch mononuclear cells using the magnetic-activated cell sorting system. The expression of IDO mRNA was analyzed using RT-PCR. β 2 -Actin was used as an internal control. Each value is the mean ± standard deviation of replicate determinations in one of four experiments. * P
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    Vanillic acid inhibits the proliferation of HCT116 cells via blocking cell cycle progression in the G1 phase. ( A ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Subsequently, cell cycle status was analyzed by by FACSCalibur flow cytometry. ( B ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Cyclin D1 and c-Myc were detected by Western blot analysis. Levels of tubulin were used as a loading control. ( C ) Data were shown as mean ± SD ( n = 3). ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Vanillic Acid Suppresses HIF-1α Expression via Inhibition of mTOR/p70S6K/4E-BP1 and Raf/MEK/ERK Pathways in Human Colon Cancer HCT116 Cells

    doi: 10.3390/ijms20030465

    Figure Lengend Snippet: Vanillic acid inhibits the proliferation of HCT116 cells via blocking cell cycle progression in the G1 phase. ( A ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Subsequently, cell cycle status was analyzed by by FACSCalibur flow cytometry. ( B ) HCT116 cells were pretreated without or with indicated concentration of vanillic acid (Van), then cultured under normoxic or hypoxic conditions for 12 h. Cyclin D1 and c-Myc were detected by Western blot analysis. Levels of tubulin were used as a loading control. ( C ) Data were shown as mean ± SD ( n = 3). ** p

    Article Snippet: The DNA content was analyzed using a FACSCalibur flow cytometer with Cell Quest software (Becton-Dickinson, Franklin Lakes, NJ, USA).

    Techniques: Blocking Assay, Concentration Assay, Cell Culture, Flow Cytometry, Cytometry, Western Blot

    CD8 T cells responding to lytic (ORF65 131–140 /D d ) or latent (M2 91–99 /K d ) Ags have different abilities to kill peptide-loaded targets in vivo. Latently infected BALB/c mice ( > 40 days postinfection with MHV-68) were given 2 × 10 7 fluorescently labeled splenocytes i.v. as described in Materials and Methods . In the first experiment ( A ) killing was analyzed 16 or 40 h later, whereas in the second experiment ( B ) killing was analyzed 40 or 64 h postinjection. To measure Ag-specific killing, splenocytes were harvested, incubated with 20 µg/ml 7-AAD for 15 min and then analyzed using a FACSCalibur cytometer. Specific lysis was then determined as described in Materials and Methods and the killing of M2 91–99 (○) and ORF65 131–140 (△) labeled cells is shown in the graph. Each symbol represents one mouse with the bar indicating the average for that group. p values for a given time-point are and were determined using Student’s t test.

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

    Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection

    doi:

    Figure Lengend Snippet: CD8 T cells responding to lytic (ORF65 131–140 /D d ) or latent (M2 91–99 /K d ) Ags have different abilities to kill peptide-loaded targets in vivo. Latently infected BALB/c mice ( > 40 days postinfection with MHV-68) were given 2 × 10 7 fluorescently labeled splenocytes i.v. as described in Materials and Methods . In the first experiment ( A ) killing was analyzed 16 or 40 h later, whereas in the second experiment ( B ) killing was analyzed 40 or 64 h postinjection. To measure Ag-specific killing, splenocytes were harvested, incubated with 20 µg/ml 7-AAD for 15 min and then analyzed using a FACSCalibur cytometer. Specific lysis was then determined as described in Materials and Methods and the killing of M2 91–99 (○) and ORF65 131–140 (△) labeled cells is shown in the graph. Each symbol represents one mouse with the bar indicating the average for that group. p values for a given time-point are and were determined using Student’s t test.

    Article Snippet: Cells were incubated with anti-CD16/CD32 Fc block (BD PharMingen) for 10 min on ice; staining with tetrameric reagents took place for 1 h at room temperature, followed by staining with anti-CD8a PerCP (clone 53-6.7) and anti-CD62L FITC (clone MEL-14) or anti-CD43 PE (clone 1B11) on ice for 20 min. Stained samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Immunocytometry Systems, Mountain View, CA).

    Techniques: In Vivo, Infection, Mouse Assay, Labeling, Incubation, Cytometry, Lysis

    Survival of lytic or latent Ag-specific CD8 T cells in response to IL-7 or IL-15 in vitro. Spleen cells from BALB/c mice infected for > 40 days with MHV-68 were labeled with CFSE then placed in culture with 25 ng/ml recombinant mouse IL-15 or 25 ng/ml recombinant mouse IL-7. Four days later, the cells were harvested, counted, and stained with tetramers consisting of the M2 91–99 /K d epitope (○), or the ORF65 131–140 /D d epitope (●) and anti-CD8a Ab. To identify dead cells, samples were incubated with 20 µg/ml 7-AAD for 15 min before analyzing on a FACSCalibur cytometer. A , Gating used for analysis of the data. B , The number of live tetramer-positive cells in the different treatment groups relative to the numbers obtained after culture with medium alone. Each point represents data from a different experiment, a total of six experiments were performed. Values for p were calculated using Student’s t test.

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

    Article Title: Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection

    doi:

    Figure Lengend Snippet: Survival of lytic or latent Ag-specific CD8 T cells in response to IL-7 or IL-15 in vitro. Spleen cells from BALB/c mice infected for > 40 days with MHV-68 were labeled with CFSE then placed in culture with 25 ng/ml recombinant mouse IL-15 or 25 ng/ml recombinant mouse IL-7. Four days later, the cells were harvested, counted, and stained with tetramers consisting of the M2 91–99 /K d epitope (○), or the ORF65 131–140 /D d epitope (●) and anti-CD8a Ab. To identify dead cells, samples were incubated with 20 µg/ml 7-AAD for 15 min before analyzing on a FACSCalibur cytometer. A , Gating used for analysis of the data. B , The number of live tetramer-positive cells in the different treatment groups relative to the numbers obtained after culture with medium alone. Each point represents data from a different experiment, a total of six experiments were performed. Values for p were calculated using Student’s t test.

    Article Snippet: Cells were incubated with anti-CD16/CD32 Fc block (BD PharMingen) for 10 min on ice; staining with tetrameric reagents took place for 1 h at room temperature, followed by staining with anti-CD8a PerCP (clone 53-6.7) and anti-CD62L FITC (clone MEL-14) or anti-CD43 PE (clone 1B11) on ice for 20 min. Stained samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Immunocytometry Systems, Mountain View, CA).

    Techniques: In Vitro, Mouse Assay, Infection, Labeling, Recombinant, Staining, Incubation, Cytometry

    Protein and gene expression of components of HS biosynthesis in presence of heparin. (A) Protein expression of HS-modifying enzymes (NDST1, C5-epimerase, HS2ST and HS3ST5) in transfected cells previously treated with heparin was evaluated by flow cytometry using antibodies specific for each enzyme. Following incubation with primary antibodies, cells were incubated with secondary antibody conjugated with Alexa Fluor® 633 and analyzed on FACSCalibur flow cytometer. (B) PAPS synthases mRNA level in EC-HS3ST5 cells treated with heparin was analyzed by real-time. The results were expressed as mean ± standard deviation. (Right panel) Heat map was generated of mean values obtained in the gene expression assays. High and low expression are shown in red and blue colors. *, Differences statistically significant, P

    Journal: bioRxiv

    Article Title: Heparan sulfate structure is influenced by the ER-Golgi dynamics of its modifying enzymes

    doi: 10.1101/2020.01.23.916940

    Figure Lengend Snippet: Protein and gene expression of components of HS biosynthesis in presence of heparin. (A) Protein expression of HS-modifying enzymes (NDST1, C5-epimerase, HS2ST and HS3ST5) in transfected cells previously treated with heparin was evaluated by flow cytometry using antibodies specific for each enzyme. Following incubation with primary antibodies, cells were incubated with secondary antibody conjugated with Alexa Fluor® 633 and analyzed on FACSCalibur flow cytometer. (B) PAPS synthases mRNA level in EC-HS3ST5 cells treated with heparin was analyzed by real-time. The results were expressed as mean ± standard deviation. (Right panel) Heat map was generated of mean values obtained in the gene expression assays. High and low expression are shown in red and blue colors. *, Differences statistically significant, P

    Article Snippet: Data were collected in the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA) and data analysis were performed using FlowJo v.10 software (Tree Star Inc, Ashand, USA).

    Techniques: Expressing, Transfection, Flow Cytometry, Incubation, Papanicolaou Stain, Standard Deviation, Generated

    Oral tolerance induction in indoleamine 2,3-dioxygenase-expressing CD11c + dendritic cells of tolerized mice. The induction of oral tolerance increases the proportion of indoleamine 2,3-dioxygenase (IDO)-expressing CD11c + dendritic cells (DCs) in Peyer's patches of tolerized mice. (a) Flow cytometric analysis of IDO in CD11c + DCs isolated from Peyer's patches. Mononuclear cells obtained from Peyer's patches of tolerized mice and of CIA mice were probed with Fluorescein isothiocyanate-labeled anti-CD11c mAb and were fixed with CytoPerm/CytoFix for 20 minutes. Cells were probed for intracellular IDO using anti-mouse IDO antibody and were analyzed by flow cytometry. The histograms were gated on CD11c + DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Results are the mean ± standard deviation of replicate samples from seven independent experiments. SSC. (b) Analysis of IDO transcription in tolerized mice and CIA mice. CD11c + DCs were isolated from Peyer's patch mononuclear cells using the magnetic-activated cell sorting system. The expression of IDO mRNA was analyzed using RT-PCR. β 2 -Actin was used as an internal control. Each value is the mean ± standard deviation of replicate determinations in one of four experiments. * P

    Journal: Arthritis Research & Therapy

    Article Title: Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model

    doi: 10.1186/ar2361

    Figure Lengend Snippet: Oral tolerance induction in indoleamine 2,3-dioxygenase-expressing CD11c + dendritic cells of tolerized mice. The induction of oral tolerance increases the proportion of indoleamine 2,3-dioxygenase (IDO)-expressing CD11c + dendritic cells (DCs) in Peyer's patches of tolerized mice. (a) Flow cytometric analysis of IDO in CD11c + DCs isolated from Peyer's patches. Mononuclear cells obtained from Peyer's patches of tolerized mice and of CIA mice were probed with Fluorescein isothiocyanate-labeled anti-CD11c mAb and were fixed with CytoPerm/CytoFix for 20 minutes. Cells were probed for intracellular IDO using anti-mouse IDO antibody and were analyzed by flow cytometry. The histograms were gated on CD11c + DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Results are the mean ± standard deviation of replicate samples from seven independent experiments. SSC. (b) Analysis of IDO transcription in tolerized mice and CIA mice. CD11c + DCs were isolated from Peyer's patch mononuclear cells using the magnetic-activated cell sorting system. The expression of IDO mRNA was analyzed using RT-PCR. β 2 -Actin was used as an internal control. Each value is the mean ± standard deviation of replicate determinations in one of four experiments. * P

    Article Snippet: Cells were stained further with rabbit anti-IDO polyclonal antibody (Transgenic Inc, Kobe, Japan), followed by Phycoerythrin-conjugated goat anti-rabbit immunoglobulin, and then subjected to flow cytometric analysis (FACSCalibur; Becton Dickinson, San Jose, CA, USA).

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Isolation, Labeling, Cytometry, Staining, Standard Deviation, FACS, Reverse Transcription Polymerase Chain Reaction