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BioLegend cd45
Inducing effct of lymphocytes and autophagy inhibitors on the expression of PD-L1 by gastric cancer cell lines. a Gating strategy of the cocultures. Gastric cancer cells were gated according to morphology (FSC-A vs. SSC-A) to single cell discrimination (SSC-W vs. SSC-A). The gastric cancer cells were then gated to perform live/dead and lymphocyte discrimination <t>(CD45</t> vs. 7AAD). These cells were then checked for PD-L1 positivity (PD-L1 vs. 7AAD). The assays on each single sample were repeated at least 3 times. b Evaluation of the expression of PD-L1 in the gastric cancer cell lines AGS or NCI-n87 cocultured with lymphocytes in the presence of chloroquine or 3-MA. Control cells was off drugs for 3 days before the harvest for flowcytometry (Material and Methods). The ratio of PD-L1 MFI minus Isotype control was shown as mean ± S.D. relative to Ctrl from 3 independent experiments, * p
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1) Product Images from "Autophagy inhibition enhances PD-L1 expression in gastric cancer"

Article Title: Autophagy inhibition enhances PD-L1 expression in gastric cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-019-1148-5

Inducing effct of lymphocytes and autophagy inhibitors on the expression of PD-L1 by gastric cancer cell lines. a Gating strategy of the cocultures. Gastric cancer cells were gated according to morphology (FSC-A vs. SSC-A) to single cell discrimination (SSC-W vs. SSC-A). The gastric cancer cells were then gated to perform live/dead and lymphocyte discrimination (CD45 vs. 7AAD). These cells were then checked for PD-L1 positivity (PD-L1 vs. 7AAD). The assays on each single sample were repeated at least 3 times. b Evaluation of the expression of PD-L1 in the gastric cancer cell lines AGS or NCI-n87 cocultured with lymphocytes in the presence of chloroquine or 3-MA. Control cells was off drugs for 3 days before the harvest for flowcytometry (Material and Methods). The ratio of PD-L1 MFI minus Isotype control was shown as mean ± S.D. relative to Ctrl from 3 independent experiments, * p
Figure Legend Snippet: Inducing effct of lymphocytes and autophagy inhibitors on the expression of PD-L1 by gastric cancer cell lines. a Gating strategy of the cocultures. Gastric cancer cells were gated according to morphology (FSC-A vs. SSC-A) to single cell discrimination (SSC-W vs. SSC-A). The gastric cancer cells were then gated to perform live/dead and lymphocyte discrimination (CD45 vs. 7AAD). These cells were then checked for PD-L1 positivity (PD-L1 vs. 7AAD). The assays on each single sample were repeated at least 3 times. b Evaluation of the expression of PD-L1 in the gastric cancer cell lines AGS or NCI-n87 cocultured with lymphocytes in the presence of chloroquine or 3-MA. Control cells was off drugs for 3 days before the harvest for flowcytometry (Material and Methods). The ratio of PD-L1 MFI minus Isotype control was shown as mean ± S.D. relative to Ctrl from 3 independent experiments, * p

Techniques Used: Expressing

2) Product Images from "A Study of Zoledronic Acid as Neo-Adjuvant, Perioperative Therapy in Patients with Resectable Pancreatic Ductal Adenocarcinoma"

Article Title: A Study of Zoledronic Acid as Neo-Adjuvant, Perioperative Therapy in Patients with Resectable Pancreatic Ductal Adenocarcinoma

Journal: Journal of cancer therapy

doi: 10.4236/jct.2013.43096

Peripheral blood and bone marrow mononuclear cells from PDAC patients were collected pre- and post-treatment with zoledronic acid (ZA) and flow cytometry was performed to compare changes in the prevalence of myeloid derived suppressor cells (CD45+, Lin−, CD33+, CD11b+, CD15+). (a) Representative flow cytometry plot from blood of PDAC patient. (b) Graph depicts G-MDSC prevalence in the blood pre- and post-treatment. (c) Representative flow cytometry plot from bone marrow of PDAC patient. (d) Graph depicts G-MDSC prevalence in the bone marrow of PDAC patients pre- and post-treatment with ZA. Graphs depict means ± SEM. p values are by paired t-tests.
Figure Legend Snippet: Peripheral blood and bone marrow mononuclear cells from PDAC patients were collected pre- and post-treatment with zoledronic acid (ZA) and flow cytometry was performed to compare changes in the prevalence of myeloid derived suppressor cells (CD45+, Lin−, CD33+, CD11b+, CD15+). (a) Representative flow cytometry plot from blood of PDAC patient. (b) Graph depicts G-MDSC prevalence in the blood pre- and post-treatment. (c) Representative flow cytometry plot from bone marrow of PDAC patient. (d) Graph depicts G-MDSC prevalence in the bone marrow of PDAC patients pre- and post-treatment with ZA. Graphs depict means ± SEM. p values are by paired t-tests.

Techniques Used: Flow Cytometry, Cytometry, Derivative Assay

3) Product Images from "Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans"

Article Title: Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

Journal: bioRxiv

doi: 10.1101/2020.01.03.894709

Titanizaiton and disease outcomes on mice model (A) Cell body sizes of fungal cells collected from murine lungs (n > 70 cells for each isolate). (B, C) Infectious burdens for the indicated strains. Murine lungs (B) and brains (C) were assessed for dissemination 10 days post-infection via colony forming units of total homogenates (n=5 per strain). Data are presented as Log CFU/gram. (B) Data were assessed as normal by Shapiro-Wilk and analysed by One-Way ANOVA with Holm-Sidak’s multiple comparisons test. Data are presented as Log CFU/gram. (C) Data were assessed as not normal by Shapiro-Wilk and analysed by Kruskal-Wallis ANOVA with Dunn’s correction for multiple comparisons. (D, E) Immune cell recruitment to lungs for the indicated strains. Lung homogenates were analysed by FACS for the indicated cell populations identified using the indicated markers for (D) Neutrophils (CD45+, CD11b+Ly6G+) or (E) Alveolar Macrophages (CD45+ Ly6C-Ly6G-CD11b-SiglecF+). Data were assessed as normal by Shapiro-Wilk and analysed by One-Way ANOVA with Holm-Sidak’s multiple comparisons test.
Figure Legend Snippet: Titanizaiton and disease outcomes on mice model (A) Cell body sizes of fungal cells collected from murine lungs (n > 70 cells for each isolate). (B, C) Infectious burdens for the indicated strains. Murine lungs (B) and brains (C) were assessed for dissemination 10 days post-infection via colony forming units of total homogenates (n=5 per strain). Data are presented as Log CFU/gram. (B) Data were assessed as normal by Shapiro-Wilk and analysed by One-Way ANOVA with Holm-Sidak’s multiple comparisons test. Data are presented as Log CFU/gram. (C) Data were assessed as not normal by Shapiro-Wilk and analysed by Kruskal-Wallis ANOVA with Dunn’s correction for multiple comparisons. (D, E) Immune cell recruitment to lungs for the indicated strains. Lung homogenates were analysed by FACS for the indicated cell populations identified using the indicated markers for (D) Neutrophils (CD45+, CD11b+Ly6G+) or (E) Alveolar Macrophages (CD45+ Ly6C-Ly6G-CD11b-SiglecF+). Data were assessed as normal by Shapiro-Wilk and analysed by One-Way ANOVA with Holm-Sidak’s multiple comparisons test.

Techniques Used: Mouse Assay, Infection, FACS

4) Product Images from "Contribution of dermal-derived mesenchymal cells during liver repair in two different experimental models"

Article Title: Contribution of dermal-derived mesenchymal cells during liver repair in two different experimental models

Journal: Scientific Reports

doi: 10.1038/srep25314

Identification of engraftment DMCs in liver. To further explore what phenotypes the GFP positive cells exhibited, donor dermal cells in the liver of transplanted mice were investigated by immunofluorescence. All GFP positive cells engrafted in the liver were labeled with vimentin, which implied that they were originated from dermis ( A ). As followed, to detect whether these cells displayed markers of liver cells, we incubated them with markers of liver progenitor cells (PCK), sinusoid endothelial cells (VEGFR2), and hepatic stellate cells (αSMA) respectively and the results showed none of these molecules expressed ( B–D ). However, all GFP positive cells presented the hematopoietic marker CD45 and macrophage surface maker F4/80 ( E,F ). Colocalization (arrows) of the GFP (green) with vimintin ( A ), CD45 ( E ) and F4/80 ( F ) staining respectively indicates that DMCs were expressing vimintin, CD45 and F4/80.Bar represent 50 μm (400× magnification).
Figure Legend Snippet: Identification of engraftment DMCs in liver. To further explore what phenotypes the GFP positive cells exhibited, donor dermal cells in the liver of transplanted mice were investigated by immunofluorescence. All GFP positive cells engrafted in the liver were labeled with vimentin, which implied that they were originated from dermis ( A ). As followed, to detect whether these cells displayed markers of liver cells, we incubated them with markers of liver progenitor cells (PCK), sinusoid endothelial cells (VEGFR2), and hepatic stellate cells (αSMA) respectively and the results showed none of these molecules expressed ( B–D ). However, all GFP positive cells presented the hematopoietic marker CD45 and macrophage surface maker F4/80 ( E,F ). Colocalization (arrows) of the GFP (green) with vimintin ( A ), CD45 ( E ) and F4/80 ( F ) staining respectively indicates that DMCs were expressing vimintin, CD45 and F4/80.Bar represent 50 μm (400× magnification).

Techniques Used: Mouse Assay, Immunofluorescence, Labeling, Incubation, Marker, Staining, Expressing

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-020-64685-y

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

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

6) Product Images from "Incomplete Deletion of IL-4Rα by LysMCre Reveals Distinct Subsets of M2 Macrophages Controlling Inflammation and Fibrosis in Chronic Schistosomiasis"

Article Title: Incomplete Deletion of IL-4Rα by LysMCre Reveals Distinct Subsets of M2 Macrophages Controlling Inflammation and Fibrosis in Chronic Schistosomiasis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004372

Macrophage populations in livers of S. mansoni -infected IL-4Rα flox/Δ LysM Cre mice express Il4r α and alternative activation markers. IL-4Rα flox/Δ LysM Cre mice (open bars) and IL-4Rα flox/Δ littermate controls (solid bars) were infected percutaneously with 35 cercariae. From mice infected for 9 weeks, CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ liver leukocytes were sorted and separated based on Ly6C expression with a flow cytometer. Gene expression was measured by qPCR (n = 3; *p
Figure Legend Snippet: Macrophage populations in livers of S. mansoni -infected IL-4Rα flox/Δ LysM Cre mice express Il4r α and alternative activation markers. IL-4Rα flox/Δ LysM Cre mice (open bars) and IL-4Rα flox/Δ littermate controls (solid bars) were infected percutaneously with 35 cercariae. From mice infected for 9 weeks, CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ liver leukocytes were sorted and separated based on Ly6C expression with a flow cytometer. Gene expression was measured by qPCR (n = 3; *p

Techniques Used: Infection, Mouse Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

7) Product Images from "The RNA binding protein SORBS2 suppresses metastatic colonization of ovarian cancer by stabilizing tumor-suppressive immunomodulatory transcripts"

Article Title: The RNA binding protein SORBS2 suppresses metastatic colonization of ovarian cancer by stabilizing tumor-suppressive immunomodulatory transcripts

Journal: Genome Biology

doi: 10.1186/s13059-018-1412-6

Cancer-derived SORBS2-stabilized secretome suppresses tumor metastasis and recruitment of tumor-supportive infiltrates in vivo. a The percentage of CD11b + GR-1+ cells in the CD45+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and WFDC1 overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. b The percentage of CD11b + GR-1+ cells in the CD45+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and IL-17D-overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. c The percentage of CD206+ cells in the CD11b + GR-1+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and WFDC1-overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. d The percentage of CD206+ cells in the CD11b + GR-1+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and IL-17D-overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. e At the global level, SORBS2 could bind different kinds of mRNAs and stabilize a proportion of these mRNAs. The net effect of progression-promoting and -inhibiting alterations determine whether SORBS2 loss is beneficial for ovarian cancer metastatic colonization. f At the target mRNA level, SORBS2 could stabilize the transcripts of WFDC1 and IL-17D, which leads to overexpression of these secreting factors. On one hand, they could partly suppress ovarian cancer metastasis; on the other hand, they could inhibit the polarization of monocytes towards MDSCs and M2-like macrophages, which is important for a immune suppressive tumor microenvironment favorable for ovarian cancer metastatic colonization. Data are shown as mean ± SEM. * P
Figure Legend Snippet: Cancer-derived SORBS2-stabilized secretome suppresses tumor metastasis and recruitment of tumor-supportive infiltrates in vivo. a The percentage of CD11b + GR-1+ cells in the CD45+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and WFDC1 overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. b The percentage of CD11b + GR-1+ cells in the CD45+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and IL-17D-overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. c The percentage of CD206+ cells in the CD11b + GR-1+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and WFDC1-overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. d The percentage of CD206+ cells in the CD11b + GR-1+ cells of the metastatic nodules of C57BL/6 mice intrabursally inoculated with control ID-8 cells, SORBS2-knockdown ID-8 cells, and IL-17D-overexpressing SORBS2-knockdown ID-8 cells. n = 6 in each group. e At the global level, SORBS2 could bind different kinds of mRNAs and stabilize a proportion of these mRNAs. The net effect of progression-promoting and -inhibiting alterations determine whether SORBS2 loss is beneficial for ovarian cancer metastatic colonization. f At the target mRNA level, SORBS2 could stabilize the transcripts of WFDC1 and IL-17D, which leads to overexpression of these secreting factors. On one hand, they could partly suppress ovarian cancer metastasis; on the other hand, they could inhibit the polarization of monocytes towards MDSCs and M2-like macrophages, which is important for a immune suppressive tumor microenvironment favorable for ovarian cancer metastatic colonization. Data are shown as mean ± SEM. * P

Techniques Used: Derivative Assay, In Vivo, Mouse Assay, Over Expression

8) Product Images from "BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s reveal distinct pathophysiologic features and impact on clinical outcomes in melanoma patients"

Article Title: BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s reveal distinct pathophysiologic features and impact on clinical outcomes in melanoma patients

Journal: Clinical & Translational Immunology

doi: 10.1002/cti2.1190

Decreased frequencies of circulating DC subsets in melanoma patients and infiltration level of the tumor site determine the clinical outcome of patients. PBMC and tumor‐infiltrating cells from melanoma patients together with PBMC from HD and control tissues were labelled with specific antibodies allowing depicting the three DC subsets and submitted to flow cytometry analysis. (a) Comparative frequencies of BDCA1 + cDC2s, BDCA2 + pDCs and BDCA3 + cDC1s within alive CD45 + cells on the blood of healthy donors (HD, open circles, n = 56 to 67) and patients (Pt, filled circles, n = 17), non‐tumor tissue (tonsils, open triangles, n = 9) and tumor infiltrate of melanoma patients (filled triangles, n = 23). Results are expressed as percentages of positive cells. Bars indicate mean. P ‐values were calculated using Mann–Whitney (dashed lines) and Kruskal–Wallis (straight lines) nonparametric tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. (b) Relative proportions of each DC subsets within all DCs in patients’ blood ( n = 17) and tumor infiltrates ( n = 23). Bars indicate mean. P ‐values were calculated using the Mann–Whitney test. (c) Correlation matrix between the three DC subsets frequencies in HD blood (left panel), patient blood (middle panel) and tumor infiltrate (right panel). Spearman correlations r factors with their significant P ‐values (
Figure Legend Snippet: Decreased frequencies of circulating DC subsets in melanoma patients and infiltration level of the tumor site determine the clinical outcome of patients. PBMC and tumor‐infiltrating cells from melanoma patients together with PBMC from HD and control tissues were labelled with specific antibodies allowing depicting the three DC subsets and submitted to flow cytometry analysis. (a) Comparative frequencies of BDCA1 + cDC2s, BDCA2 + pDCs and BDCA3 + cDC1s within alive CD45 + cells on the blood of healthy donors (HD, open circles, n = 56 to 67) and patients (Pt, filled circles, n = 17), non‐tumor tissue (tonsils, open triangles, n = 9) and tumor infiltrate of melanoma patients (filled triangles, n = 23). Results are expressed as percentages of positive cells. Bars indicate mean. P ‐values were calculated using Mann–Whitney (dashed lines) and Kruskal–Wallis (straight lines) nonparametric tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. (b) Relative proportions of each DC subsets within all DCs in patients’ blood ( n = 17) and tumor infiltrates ( n = 23). Bars indicate mean. P ‐values were calculated using the Mann–Whitney test. (c) Correlation matrix between the three DC subsets frequencies in HD blood (left panel), patient blood (middle panel) and tumor infiltrate (right panel). Spearman correlations r factors with their significant P ‐values (

Techniques Used: Flow Cytometry, MANN-WHITNEY

9) Product Images from "Thymopentin ameliorates dextran sulfate sodium-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes"

Article Title: Thymopentin ameliorates dextran sulfate sodium-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes

Journal: Theranostics

doi: 10.7150/thno.35015

TP5 promoted IL-22 expression via RORγt. (A) RT-PCR results of stat3, Ahr, RORγt, and NF-AT in mouse colons. (n=4-5). (B-C) Flow cytometry analysis of ILC3s (CD45 + , CD4 - , RORγt + ) and CD4 + RORγt + cells (CD45 + , CD4 + , RORγt + ) in colon LPMCs. (n=4-5). # P
Figure Legend Snippet: TP5 promoted IL-22 expression via RORγt. (A) RT-PCR results of stat3, Ahr, RORγt, and NF-AT in mouse colons. (n=4-5). (B-C) Flow cytometry analysis of ILC3s (CD45 + , CD4 - , RORγt + ) and CD4 + RORγt + cells (CD45 + , CD4 + , RORγt + ) in colon LPMCs. (n=4-5). # P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry

TP5 restored circulating lymphocytes. (A) Blood examination of mice, including WBC, RBC, PLT, and percentage of NEU, LYM, MONO, EOS, BASO, and LUC. (n=4-5). (B) Thymus coefficient, spleen coefficient and lymph node coefficient in mice. (n=9-10). Organ (Thymus/spleen/lymph node) coefficient = Organ (Thymus/ spleen/lymph node) weight/body weight × 100. (C) Flow cytometry analysis of double positive (DP) cells (CD45 + , CD4 + , CD8 + ) in the thymus. (n=4-5). (D) Representative H E staining photographs of the thymus in each group. The arrows showed the necrotic cells. The scale bar represents 50 μm. (n=4-5). (E) Flow cytometry analysis of T cells (CD45 + , CD3e + ) in the spleen. (n=4-5). * P
Figure Legend Snippet: TP5 restored circulating lymphocytes. (A) Blood examination of mice, including WBC, RBC, PLT, and percentage of NEU, LYM, MONO, EOS, BASO, and LUC. (n=4-5). (B) Thymus coefficient, spleen coefficient and lymph node coefficient in mice. (n=9-10). Organ (Thymus/spleen/lymph node) coefficient = Organ (Thymus/ spleen/lymph node) weight/body weight × 100. (C) Flow cytometry analysis of double positive (DP) cells (CD45 + , CD4 + , CD8 + ) in the thymus. (n=4-5). (D) Representative H E staining photographs of the thymus in each group. The arrows showed the necrotic cells. The scale bar represents 50 μm. (n=4-5). (E) Flow cytometry analysis of T cells (CD45 + , CD3e + ) in the spleen. (n=4-5). * P

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

TP5 diminished inflammation in the colon and increased IL-22 expression. (A) RT-PCR results of IL-6 , IL-1β , IL-1α , TNF-α , IL-18 , IFN-γ , and CCL-2 in colons of mice. (n=4-5). (B) Representative F4/80 staining photographs, and mean F4/80 positive cells counted in 5 fields. The scale bar represents 50 μm. (n=4-5). (C) RT-PCR results of IL-10 , IL-22 , IL-23 , IL-12 , and TGF-β in mouse colons. (n=4-5). (D) ELISA result of IL-22 in mouse colons and blood. (n=4-5). (E) Flow cytometry analysis of IL-22-producing splenocytes (CD45 + , CD4 + , IL-22 + ) treated with TP5 in vitro . (n=4). (F) RT-PCR results of IL-22 expression induced in splenocytes by TP5 in vitro . (n=4). (G) Flow cytometry analysis of IL-22-producing ILC3s (CD45 + , CD4 - , RORγt + , IL-22 + ) LPMCs treated with TP5 in vitro . (n=4). * P
Figure Legend Snippet: TP5 diminished inflammation in the colon and increased IL-22 expression. (A) RT-PCR results of IL-6 , IL-1β , IL-1α , TNF-α , IL-18 , IFN-γ , and CCL-2 in colons of mice. (n=4-5). (B) Representative F4/80 staining photographs, and mean F4/80 positive cells counted in 5 fields. The scale bar represents 50 μm. (n=4-5). (C) RT-PCR results of IL-10 , IL-22 , IL-23 , IL-12 , and TGF-β in mouse colons. (n=4-5). (D) ELISA result of IL-22 in mouse colons and blood. (n=4-5). (E) Flow cytometry analysis of IL-22-producing splenocytes (CD45 + , CD4 + , IL-22 + ) treated with TP5 in vitro . (n=4). (F) RT-PCR results of IL-22 expression induced in splenocytes by TP5 in vitro . (n=4). (G) Flow cytometry analysis of IL-22-producing ILC3s (CD45 + , CD4 - , RORγt + , IL-22 + ) LPMCs treated with TP5 in vitro . (n=4). * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, In Vitro

10) Product Images from "Persistent Lung Inflammation and Fibrosis in Serum Amyloid P Component (Apcs-/-) Knockout Mice"

Article Title: Persistent Lung Inflammation and Fibrosis in Serum Amyloid P Component (Apcs-/-) Knockout Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093730

Changes in tissue leukocyte population in C57BL/6 and Apcs -/- mice following bleomycin instillation. Cryosections of mouse lungs were labeled with antibodies against A ) CD11b (neutrophils and inflammatory macrophages), B ) CD11c (resident macrophages and dendritic cells), C ) CD45 (total leukocytes), D ) CD163 (macrophages), E ) CD206 (macrophages), and F ) Ly6G (neutrophils). Values are mean ± SEM (n = 4–6 mice per group). Significance was determined by ANOVA within treatment groups and t-test when comparing C57BL/6 and Apcs -/- mice.
Figure Legend Snippet: Changes in tissue leukocyte population in C57BL/6 and Apcs -/- mice following bleomycin instillation. Cryosections of mouse lungs were labeled with antibodies against A ) CD11b (neutrophils and inflammatory macrophages), B ) CD11c (resident macrophages and dendritic cells), C ) CD45 (total leukocytes), D ) CD163 (macrophages), E ) CD206 (macrophages), and F ) Ly6G (neutrophils). Values are mean ± SEM (n = 4–6 mice per group). Significance was determined by ANOVA within treatment groups and t-test when comparing C57BL/6 and Apcs -/- mice.

Techniques Used: Mouse Assay, Labeling

11) Product Images from "Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils"

Article Title: Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils

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

doi: 10.1126/science.aal5081

Lung tumors increase osteoblast activity in mice. ( A . ( B ) Number of osteoblasts per bone surface in distal femur trabecular bone from the same mice as in (A) (n = 4 mice per group). ( C ) Flow cytometry-based quantification of the percentage of bone marrow Ocn-YFP + cells isolated from tumor-free mice and KP lung tumor-bearing Ocn Cre;Yfp mice (n = 6 mice per group). Ocn-YFP + cells were defined as 7AAD − Lin − CD45 − CD31 − Ter119 − YFP + . ( D ) Representative von Kossa staining (left) and quantification of mineralized bone (% von Kossa area, right) in femurs from the same mice as in (A) (n = 4 mice per group). Scale bar 1 mm. ( E for additional measurements. All figures show mean ± SEM. Statistical significance was calculated using an unpaired t-test. * p
Figure Legend Snippet: Lung tumors increase osteoblast activity in mice. ( A . ( B ) Number of osteoblasts per bone surface in distal femur trabecular bone from the same mice as in (A) (n = 4 mice per group). ( C ) Flow cytometry-based quantification of the percentage of bone marrow Ocn-YFP + cells isolated from tumor-free mice and KP lung tumor-bearing Ocn Cre;Yfp mice (n = 6 mice per group). Ocn-YFP + cells were defined as 7AAD − Lin − CD45 − CD31 − Ter119 − YFP + . ( D ) Representative von Kossa staining (left) and quantification of mineralized bone (% von Kossa area, right) in femurs from the same mice as in (A) (n = 4 mice per group). Scale bar 1 mm. ( E for additional measurements. All figures show mean ± SEM. Statistical significance was calculated using an unpaired t-test. * p

Techniques Used: Activity Assay, Mouse Assay, Flow Cytometry, Cytometry, Isolation, Staining

Ocn + cell-driven neutrophils show discrete phenotypes. ( A ) Flow cytometry-based detection (left) of Ly-6G + SiglecF high or low neutrophils from healthy lung tissue (top) and KP1.9 lung tumors (bottom). Plots are shown for gated live CD45 + CD11b + cells. Representative cytospin images (right) are from FACS-sorted populations further stained with H E. Scale bar 10 μm. ( B ) Fold change Ly-6G + SiglecF high and Ly-6G + SiglecF low cell number in lungs from tumor bearing-mice when compared to tumor-free mice. Cells were assessed by flow cytometry (n = 6 mice per group). ( C ) Representative SiglecF mAb staining on cryo-preserved KP lung tumor tissue. Tumor areas are highlighted by dotted purple lines. Scale bar 50 μm. ( D ) Flow cytometry-based quantification of Ly-6G + SiglecF high and Ly-6G + SiglecF low cells in tumor-bearing lungs of mice with either preserved Ocn + cells (pink: control mice treated with DT) or reduced numbers of these cells (green: Ocn Cre;Dtr mice treated with DT) (n = 7-9 mice per group). ( E ) Ability of CD45.1 + Lin − cKit + hematopoietic precursors to produce tumor-infiltrating SiglecF high and SiglecF low neutrophils upon transfer into KP tumor-bearing CD45.2 + recipient control mice (pink) or mice with reduced numbers of Ocn + cells (green). Mice were treated as in (D). Results are shown as fold change relative to control mice. All figures show mean ± SEM and significance values were calculated using multiple t-tests. * p
Figure Legend Snippet: Ocn + cell-driven neutrophils show discrete phenotypes. ( A ) Flow cytometry-based detection (left) of Ly-6G + SiglecF high or low neutrophils from healthy lung tissue (top) and KP1.9 lung tumors (bottom). Plots are shown for gated live CD45 + CD11b + cells. Representative cytospin images (right) are from FACS-sorted populations further stained with H E. Scale bar 10 μm. ( B ) Fold change Ly-6G + SiglecF high and Ly-6G + SiglecF low cell number in lungs from tumor bearing-mice when compared to tumor-free mice. Cells were assessed by flow cytometry (n = 6 mice per group). ( C ) Representative SiglecF mAb staining on cryo-preserved KP lung tumor tissue. Tumor areas are highlighted by dotted purple lines. Scale bar 50 μm. ( D ) Flow cytometry-based quantification of Ly-6G + SiglecF high and Ly-6G + SiglecF low cells in tumor-bearing lungs of mice with either preserved Ocn + cells (pink: control mice treated with DT) or reduced numbers of these cells (green: Ocn Cre;Dtr mice treated with DT) (n = 7-9 mice per group). ( E ) Ability of CD45.1 + Lin − cKit + hematopoietic precursors to produce tumor-infiltrating SiglecF high and SiglecF low neutrophils upon transfer into KP tumor-bearing CD45.2 + recipient control mice (pink) or mice with reduced numbers of Ocn + cells (green). Mice were treated as in (D). Results are shown as fold change relative to control mice. All figures show mean ± SEM and significance values were calculated using multiple t-tests. * p

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

12) Product Images from "Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease"

Article Title: Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease

Journal: Nature Communications

doi: 10.1038/s41467-017-02111-0

Enhanced Th1 and Th17 responses in the CNS of Bmal1 Mye−/− mice with EAE. a – e Bmal1 Myeloid+/+ or Bmal1 Myeloid−/− mice were immunized with myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) on d 0 and with pertussis toxin (PT) (125 ng/mouse) on d 0 and d 2. a After 14 d draining lymph node (LN) cells were re-stimulated in the presence and absence of MOG 35–55 antigen (25 and 50 µg/ml) or with medium for 72 h. Supernatants were removed and tested for IL-17, GM-CSF, IFN-γ and IL-10. Statistics by one way ANOVA with Tukey’s post-test of triplicate assay of 6 mice per group. b – e 10 d post immunization brain and spinal cords were isolated and surface stained for CD45, CD3, CD8α and CD4 and intracellularly for IL-17 and IFN-γ. b Cells were gated on live CD45 + cells in the brain ( n = 5–6). c Cells were gated on live CD45 + cells spinal cord ( n = 5–6). d Cells were gated on live CD45 + CD3 + cells in the brain ( n = 5–6). e Cells were gated on live CD45 + cells in the brain ( n = 5–6). Statistics were performed by Mann–Whitney U test ( b – e ). All data presented as means±standard error of the mean (SEM). * p
Figure Legend Snippet: Enhanced Th1 and Th17 responses in the CNS of Bmal1 Mye−/− mice with EAE. a – e Bmal1 Myeloid+/+ or Bmal1 Myeloid−/− mice were immunized with myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) on d 0 and with pertussis toxin (PT) (125 ng/mouse) on d 0 and d 2. a After 14 d draining lymph node (LN) cells were re-stimulated in the presence and absence of MOG 35–55 antigen (25 and 50 µg/ml) or with medium for 72 h. Supernatants were removed and tested for IL-17, GM-CSF, IFN-γ and IL-10. Statistics by one way ANOVA with Tukey’s post-test of triplicate assay of 6 mice per group. b – e 10 d post immunization brain and spinal cords were isolated and surface stained for CD45, CD3, CD8α and CD4 and intracellularly for IL-17 and IFN-γ. b Cells were gated on live CD45 + cells in the brain ( n = 5–6). c Cells were gated on live CD45 + cells spinal cord ( n = 5–6). d Cells were gated on live CD45 + CD3 + cells in the brain ( n = 5–6). e Cells were gated on live CD45 + cells in the brain ( n = 5–6). Statistics were performed by Mann–Whitney U test ( b – e ). All data presented as means±standard error of the mean (SEM). * p

Techniques Used: Mouse Assay, Isolation, Staining, MANN-WHITNEY

Enhanced accumulation of inflammatory CD11c + Ly6C + cells producing IL-1β in the CNS of Bmal1 Myeloid−/− mice during EAE. a – c Bmal1 Myeloid+/+ and Bmal1 Myeloid−/− mice were immunized to develop experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) on d 0 and with pertussis toxin (PT) (125 ng/mouse) on d 0 and d 2 ( n = 6). a D 10 post EAE induction live cells infiltrating into the spinal cord were FACS stained for CD45 ( n = 6). b Live CD45 + cells were examined for Ly6C v CD11b or CCR2 v Ly6C ( n = 6). c IL-1β expression was determined in live CD45 + CD11b + Ly6C + cells ( n = 6). d 10 d post immunization CD11b and Ly6C populations infiltrating the spinal cord of Bmal1 Myeloid−/− mice were examined for IL-1β expression by FACS, gating on live CD45 + cells ( n = 6). Data as means ±standard error of the mean (SEM). Statistics were performed by Mann–Whitney U test a – d . * p
Figure Legend Snippet: Enhanced accumulation of inflammatory CD11c + Ly6C + cells producing IL-1β in the CNS of Bmal1 Myeloid−/− mice during EAE. a – c Bmal1 Myeloid+/+ and Bmal1 Myeloid−/− mice were immunized to develop experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) on d 0 and with pertussis toxin (PT) (125 ng/mouse) on d 0 and d 2 ( n = 6). a D 10 post EAE induction live cells infiltrating into the spinal cord were FACS stained for CD45 ( n = 6). b Live CD45 + cells were examined for Ly6C v CD11b or CCR2 v Ly6C ( n = 6). c IL-1β expression was determined in live CD45 + CD11b + Ly6C + cells ( n = 6). d 10 d post immunization CD11b and Ly6C populations infiltrating the spinal cord of Bmal1 Myeloid−/− mice were examined for IL-1β expression by FACS, gating on live CD45 + cells ( n = 6). Data as means ±standard error of the mean (SEM). Statistics were performed by Mann–Whitney U test a – d . * p

Techniques Used: Mouse Assay, FACS, Staining, Expressing, MANN-WHITNEY

13) Product Images from "Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity"

Article Title: Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity

Journal: Nature immunology

doi: 10.1038/s41590-019-0421-2

Genetic ablation of TREM1 improves outcome after MCAo. a , Representative histogram of TREM1 expression in Trem1 +/+ and Trem1 −/− CD11b + CD45 + cells 2 d after MCAo. b , TREM1 expression in CD11b + CD45 hi Ly6G hi PMNs, CD11b + CD45 hi Ly6G − Mo/MΦ and CD11b + CD45 int microglia 2 d after MCAo from Trem1 +/+ , Trem1 +/− and Trem1 −/− ). c , Neurological scores after MCAo in Trem1 +/+ , Trem1 +/− and Trem1 −/− mice ( n = 9 Trem1 +/+ , n = 10 Trem1 +/− and n = 6 Trem1 −/− , mean ± s.e.m.; two-way ANOVA, repeated measures, effect of genotype +/+ versus −/−: * P = 0.0132, effect of time **** P
Figure Legend Snippet: Genetic ablation of TREM1 improves outcome after MCAo. a , Representative histogram of TREM1 expression in Trem1 +/+ and Trem1 −/− CD11b + CD45 + cells 2 d after MCAo. b , TREM1 expression in CD11b + CD45 hi Ly6G hi PMNs, CD11b + CD45 hi Ly6G − Mo/MΦ and CD11b + CD45 int microglia 2 d after MCAo from Trem1 +/+ , Trem1 +/− and Trem1 −/− ). c , Neurological scores after MCAo in Trem1 +/+ , Trem1 +/− and Trem1 −/− mice ( n = 9 Trem1 +/+ , n = 10 Trem1 +/− and n = 6 Trem1 −/− , mean ± s.e.m.; two-way ANOVA, repeated measures, effect of genotype +/+ versus −/−: * P = 0.0132, effect of time **** P

Techniques Used: Expressing, Mouse Assay

Increased gut permeability after MCAo induces TREM1 in lamina propria and blood Mo/MΦ subsets. Propranolol or vehicle was administered at reperfusion and 4 h after reperfusion, and Mo/MΦ subsets were examined in small intestine, spleen and blood at 4.5 h after reperfusion. a , Gating strategy to identify CD11b + CD45 + myeloid cells, CD11b + CD45 + Ly6G − Mo/MΦ and CD11b + CD45 + Ly6G + neutrophils in small intestine lamina propria. b , TREM1 MFI increases in the CD45 + CD11b + myeloid cell population following MCAo compared to sham control ( n = 5 sham and n = 8 biologically independent samples per group, mean ± s.e.m.; Student’s unpaired two-tailed t- test * P
Figure Legend Snippet: Increased gut permeability after MCAo induces TREM1 in lamina propria and blood Mo/MΦ subsets. Propranolol or vehicle was administered at reperfusion and 4 h after reperfusion, and Mo/MΦ subsets were examined in small intestine, spleen and blood at 4.5 h after reperfusion. a , Gating strategy to identify CD11b + CD45 + myeloid cells, CD11b + CD45 + Ly6G − Mo/MΦ and CD11b + CD45 + Ly6G + neutrophils in small intestine lamina propria. b , TREM1 MFI increases in the CD45 + CD11b + myeloid cell population following MCAo compared to sham control ( n = 5 sham and n = 8 biologically independent samples per group, mean ± s.e.m.; Student’s unpaired two-tailed t- test * P

Techniques Used: Permeability, Two Tailed Test

TREM1 is elevated in peripheral infiltrating myeloid cells early after MCAo. a , Representative plots of CD11b + CD45 hi Ly6G − Mo/MΦ and CD11b + CD45 hi Ly6G hi PMN populations at Days 0, 2 and 6 after MCAo in spleen. b , Time course of peripheral myeloid cell dynamics in spleen for 7 d after MCAo ( n = 6–8 biologically independent samples per cell type per time point, mean ± s.e.m.; one-way ANOVA per cell type; effect of PMN, P = 0.0015, effect of Mo/MΦ P = 0.06; Tukey post-hoc * P
Figure Legend Snippet: TREM1 is elevated in peripheral infiltrating myeloid cells early after MCAo. a , Representative plots of CD11b + CD45 hi Ly6G − Mo/MΦ and CD11b + CD45 hi Ly6G hi PMN populations at Days 0, 2 and 6 after MCAo in spleen. b , Time course of peripheral myeloid cell dynamics in spleen for 7 d after MCAo ( n = 6–8 biologically independent samples per cell type per time point, mean ± s.e.m.; one-way ANOVA per cell type; effect of PMN, P = 0.0015, effect of Mo/MΦ P = 0.06; Tukey post-hoc * P

Techniques Used:

TREM1 is induced on peripheral myeloid cells that infiltrate the ischemic brain. a , Diagram of the MCAo model (internal carotid artery, ICA; middle cerebral artery, MCA) carried out in 2–3-month-old male C57B6/J mice. b , Cx3cr1 GFP/+ Ccr2 RFP/+ mice were subjected to MCAo and examined for myeloid cell populations present in ischemic hemisphere 2 d later. Plots identify CD11b + CD45 hi Ly6G − Mo/MΦ, CD11b + CD45 int microglia and CD11b + CD45 hi Ly6G hi PMN. c , Representative histograms of GFP-positive CD11b + CD45 int microglia and RFP-positive CD11b + CD45 hi Ly6G − Mo/MΦ and CD11b + CD45 hi Ly6G + . d , Cx3cr1 GFP/+ Ccr2 RFP/+ mice were assayed for TREM1 surface expression in ischemic hemisphere 2 d after MCAo. Peripherally derived RFP-positive CD11b + CD45 hi Mo/MΦ and PMNs express TREM1, while CD11b + CD45 int microglia do not. e , Percentages of TREM1 + CD11b + CD45 + myeloid cells at 2 d and 6 d after MCAo in ipsilateral (IL) ischemic hemisphere relative to contralateral (CL) non-ischemic and sham IL and CL hemispheres ( n = 6 biologically independent samples per group at 2 d and n = 7 per group at 6 d, mean ± s.e.m.; two-tailed Student’s t- test ** P
Figure Legend Snippet: TREM1 is induced on peripheral myeloid cells that infiltrate the ischemic brain. a , Diagram of the MCAo model (internal carotid artery, ICA; middle cerebral artery, MCA) carried out in 2–3-month-old male C57B6/J mice. b , Cx3cr1 GFP/+ Ccr2 RFP/+ mice were subjected to MCAo and examined for myeloid cell populations present in ischemic hemisphere 2 d later. Plots identify CD11b + CD45 hi Ly6G − Mo/MΦ, CD11b + CD45 int microglia and CD11b + CD45 hi Ly6G hi PMN. c , Representative histograms of GFP-positive CD11b + CD45 int microglia and RFP-positive CD11b + CD45 hi Ly6G − Mo/MΦ and CD11b + CD45 hi Ly6G + . d , Cx3cr1 GFP/+ Ccr2 RFP/+ mice were assayed for TREM1 surface expression in ischemic hemisphere 2 d after MCAo. Peripherally derived RFP-positive CD11b + CD45 hi Mo/MΦ and PMNs express TREM1, while CD11b + CD45 int microglia do not. e , Percentages of TREM1 + CD11b + CD45 + myeloid cells at 2 d and 6 d after MCAo in ipsilateral (IL) ischemic hemisphere relative to contralateral (CL) non-ischemic and sham IL and CL hemispheres ( n = 6 biologically independent samples per group at 2 d and n = 7 per group at 6 d, mean ± s.e.m.; two-tailed Student’s t- test ** P

Techniques Used: Mouse Assay, Expressing, Derivative Assay, Two Tailed Test

14) Product Images from "Stem cells repurpose proliferation to contain a breach in their niche barrier"

Article Title: Stem cells repurpose proliferation to contain a breach in their niche barrier

Journal: eLife

doi: 10.7554/eLife.41661

HF stem cells from Cdh1 -null bulge display a gene expression profile distinct from WT HF stem cells that are naturally proliferating in anagen. ( A ) FACS strategy to purify inner bulge cells from Sox9CreER -activated HFs based on YFP + ; Sca1 - ;CD34 - ; b1 - ; CD200 Lo . ( B ) FACS strategy to purify WT bulge HF stem cells in anagen sub-stages II and/or III based on Sca1 - ; LIN - (CD45, CD140a, CD117, CD31); CD34 Hi ; a6 Hi . ( C ) Heat-map of hierarchical clustering of differentially expressed genes (p
Figure Legend Snippet: HF stem cells from Cdh1 -null bulge display a gene expression profile distinct from WT HF stem cells that are naturally proliferating in anagen. ( A ) FACS strategy to purify inner bulge cells from Sox9CreER -activated HFs based on YFP + ; Sca1 - ;CD34 - ; b1 - ; CD200 Lo . ( B ) FACS strategy to purify WT bulge HF stem cells in anagen sub-stages II and/or III based on Sca1 - ; LIN - (CD45, CD140a, CD117, CD31); CD34 Hi ; a6 Hi . ( C ) Heat-map of hierarchical clustering of differentially expressed genes (p

Techniques Used: Expressing, FACS, Hi-C

Cdh3 -Null HFs have proper architecture and undergo hair cycling normally. Scale bars, 30 μm. ( A ) FACS strategy to purify WT 2 nd telogen HF stem cells (HFSCs) for ELISA. Live cells were first gated with exclusion markers Sca1 (epidermis), CD45 (immune cells), CD140a (fibroblasts), CD31 (endothelial cells) and CD117 (c-kit + cells) [LIN-]. HF stem cells are CD34 Hi ; α6 Hi . ( B ) Concentrations of ECAD and PCAD in protein lysates of FACS-purified HF stem cells as determined by ELISA (n = 4 mice per condition/genotype). Data are mean ±SEM. *p
Figure Legend Snippet: Cdh3 -Null HFs have proper architecture and undergo hair cycling normally. Scale bars, 30 μm. ( A ) FACS strategy to purify WT 2 nd telogen HF stem cells (HFSCs) for ELISA. Live cells were first gated with exclusion markers Sca1 (epidermis), CD45 (immune cells), CD140a (fibroblasts), CD31 (endothelial cells) and CD117 (c-kit + cells) [LIN-]. HF stem cells are CD34 Hi ; α6 Hi . ( B ) Concentrations of ECAD and PCAD in protein lysates of FACS-purified HF stem cells as determined by ELISA (n = 4 mice per condition/genotype). Data are mean ±SEM. *p

Techniques Used: FACS, Enzyme-linked Immunosorbent Assay, Purification, Mouse Assay

A barrier breach within the stem cell niche results in immune cell recruitment. Scale bars, 30 μm. ( A ) CD45+ immune cells were still recruited to Cdh1 cKO bulge despite the reduced bacterial load by antibiotics treatment. ( B ) ECAD loss of function specifically in HF stem cells using Krt15CrePGR did not cause accumulation of CD45+ immune cells around cKO HF at 5 weeks post-RU, as revealed by immunofluorescence of sagittal skin sections. ( C ) ECAD loss of function in both HF stem cells and inner bulge of Krt15CrePGR -activated HFs at 4 th telogen now caused presence of CD45+ immune cell accumulation around Cdh1 -cKO bulge, as revealed by immunofluorescence of sagittal skin sections. ( D ) ECAD loss of function specifically in inner bulge of Sox9CreER -activated HFs was sufficient to recruit CD45+ immune cells. ( E ) Immunofluorescence of sagittal skin sections for immune cell markers F4/80 and CD11b. ( F, G ) Representative examples of flow cytometry analysis of various innate and adaptive immune cell populations in whole skin preps. Quantifications of data are presented in Figure 4 . Note: for solid tissues, like skin, extensive enzymatic digestion is required, which correspondingly increases the numbers of dead cells, including CD45+ immune cells ( Naik et al., 2015 ). Importantly, however, further analyses of the flow data showed that this did not alter the relative percentages of different immune cell populations in Cdh1 cKO compared to WT skins.
Figure Legend Snippet: A barrier breach within the stem cell niche results in immune cell recruitment. Scale bars, 30 μm. ( A ) CD45+ immune cells were still recruited to Cdh1 cKO bulge despite the reduced bacterial load by antibiotics treatment. ( B ) ECAD loss of function specifically in HF stem cells using Krt15CrePGR did not cause accumulation of CD45+ immune cells around cKO HF at 5 weeks post-RU, as revealed by immunofluorescence of sagittal skin sections. ( C ) ECAD loss of function in both HF stem cells and inner bulge of Krt15CrePGR -activated HFs at 4 th telogen now caused presence of CD45+ immune cell accumulation around Cdh1 -cKO bulge, as revealed by immunofluorescence of sagittal skin sections. ( D ) ECAD loss of function specifically in inner bulge of Sox9CreER -activated HFs was sufficient to recruit CD45+ immune cells. ( E ) Immunofluorescence of sagittal skin sections for immune cell markers F4/80 and CD11b. ( F, G ) Representative examples of flow cytometry analysis of various innate and adaptive immune cell populations in whole skin preps. Quantifications of data are presented in Figure 4 . Note: for solid tissues, like skin, extensive enzymatic digestion is required, which correspondingly increases the numbers of dead cells, including CD45+ immune cells ( Naik et al., 2015 ). Importantly, however, further analyses of the flow data showed that this did not alter the relative percentages of different immune cell populations in Cdh1 cKO compared to WT skins.

Techniques Used: Immunofluorescence, Flow Cytometry, Cytometry

15) Product Images from "Transcriptional profiling and single-cell chimerism analysis identifies human tissue resident T cells in the human skin after allogeneic stem cell transplantation"

Article Title: Transcriptional profiling and single-cell chimerism analysis identifies human tissue resident T cells in the human skin after allogeneic stem cell transplantation

Journal: bioRxiv

doi: 10.1101/2020.04.11.037101

Chimerism analysis of lymphocytes identifies host cells as resident cells. (a) Clinical model of allo-HSCT. Skin punch biopsy for lymphocyte isolation and chimerism analysis was performed 706 days after allo-HSCT. (b) Polymerase chain reaction of short tandem repeats (STR-PCR) of CD3 + T cells from the blood and skin and of CD45 − cells from the skin. Shown is the electropherogram of one representative locus out of 16 analyzed loci. (c) Cumulative analysis of 14 distinct detectable microsatellite loci. Shown is the median percentage of donor cells of total cells calculated from peak heights and peak areas of each of the analyzed 14 loci. (d) Single-cell genotyping through SNV using snpclust 796 days after allo-HSCT. (e) Distribution of host and donor cells identified through SNV in single-cell transcriptomes of lymphocytes from blood and skin in the allo-HSCT patient. The number inside the bar indicates the percentage of host cells within total lymphocytes (left) or total T cells (right), while the number on top indicates total lymphocytes (left) or total T cells (right) analyzed. (f) Distribution of naïve versus memory T cells within the CD4 + and CD8 + T cell lineage within host blood cells as identified by the average cluster expression of subset-specific genes. (g) Cell identity annotation was performed by average cluster expression of cell-type specific markers within host versus donor lymphocytes, which was visualized in the reduced dimensional space calculated with tSNE. (h) Genotype distribution within each of the annotated cell subsets in the skin. The number inside the black bar indicates the percentage of host cells within total cells of the respective cell subset. The number on top indicated the total cell number within the respective cell subsets.
Figure Legend Snippet: Chimerism analysis of lymphocytes identifies host cells as resident cells. (a) Clinical model of allo-HSCT. Skin punch biopsy for lymphocyte isolation and chimerism analysis was performed 706 days after allo-HSCT. (b) Polymerase chain reaction of short tandem repeats (STR-PCR) of CD3 + T cells from the blood and skin and of CD45 − cells from the skin. Shown is the electropherogram of one representative locus out of 16 analyzed loci. (c) Cumulative analysis of 14 distinct detectable microsatellite loci. Shown is the median percentage of donor cells of total cells calculated from peak heights and peak areas of each of the analyzed 14 loci. (d) Single-cell genotyping through SNV using snpclust 796 days after allo-HSCT. (e) Distribution of host and donor cells identified through SNV in single-cell transcriptomes of lymphocytes from blood and skin in the allo-HSCT patient. The number inside the bar indicates the percentage of host cells within total lymphocytes (left) or total T cells (right), while the number on top indicates total lymphocytes (left) or total T cells (right) analyzed. (f) Distribution of naïve versus memory T cells within the CD4 + and CD8 + T cell lineage within host blood cells as identified by the average cluster expression of subset-specific genes. (g) Cell identity annotation was performed by average cluster expression of cell-type specific markers within host versus donor lymphocytes, which was visualized in the reduced dimensional space calculated with tSNE. (h) Genotype distribution within each of the annotated cell subsets in the skin. The number inside the black bar indicates the percentage of host cells within total cells of the respective cell subset. The number on top indicated the total cell number within the respective cell subsets.

Techniques Used: Isolation, Polymerase Chain Reaction, Expressing

Gating strategy for isolation of lymphocytes from blood and skin. Lymphocytes were sorted as live CD45 + cells from skin and blood after enzymatic skin digestion or ficoll separation, respectively. The sorting strategy is representative for samples from the healthy donor and the allo-HSCT patient.
Figure Legend Snippet: Gating strategy for isolation of lymphocytes from blood and skin. Lymphocytes were sorted as live CD45 + cells from skin and blood after enzymatic skin digestion or ficoll separation, respectively. The sorting strategy is representative for samples from the healthy donor and the allo-HSCT patient.

Techniques Used: Isolation

Distribution of lymphocytes subsets in the skin and blood with and without allo-HSCT. (a) t-SNE depicting the cluster cell identity by different color codes based on manual annotation with known marker genes as shown in (Figure S2). scRNA-seq of fresh CD45 + lymphocytes was performed 796 days after allo-HSCT. (b) Differential distribution of lymphocyte subsets in the skin and blood of the allo-HSCT patient and a healthy control person. n.d., not determined.
Figure Legend Snippet: Distribution of lymphocytes subsets in the skin and blood with and without allo-HSCT. (a) t-SNE depicting the cluster cell identity by different color codes based on manual annotation with known marker genes as shown in (Figure S2). scRNA-seq of fresh CD45 + lymphocytes was performed 796 days after allo-HSCT. (b) Differential distribution of lymphocyte subsets in the skin and blood of the allo-HSCT patient and a healthy control person. n.d., not determined.

Techniques Used: Marker

Circulating host blood T cells display a mixed pattern of tissue specific signatures. Single-cell gene set expression scores for tissue-specific residency signatures in each host CD45 + lymphocyte detected by SNV and absence of Y-linked genes (53 cells) in the scRNA-Seq from the blood of the allo-HSCT patient. The gene set expression scores were calculated as the average expression of all genes from the set subtracted by the average expression of random background genes with similar range of expression values. The scores were obtained using the AddModuleScore function from Seurat package. Cells were clustered according to their pattern of scores for all gene sets analyzed using k-means clustering (cluster number is indicated on the right side). The numbers on the left indicate the host cell index.
Figure Legend Snippet: Circulating host blood T cells display a mixed pattern of tissue specific signatures. Single-cell gene set expression scores for tissue-specific residency signatures in each host CD45 + lymphocyte detected by SNV and absence of Y-linked genes (53 cells) in the scRNA-Seq from the blood of the allo-HSCT patient. The gene set expression scores were calculated as the average expression of all genes from the set subtracted by the average expression of random background genes with similar range of expression values. The scores were obtained using the AddModuleScore function from Seurat package. Cells were clustered according to their pattern of scores for all gene sets analyzed using k-means clustering (cluster number is indicated on the right side). The numbers on the left indicate the host cell index.

Techniques Used: Expressing

16) Product Images from "Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer"

Article Title: Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer

Journal: Nature Communications

doi: 10.1038/s41467-022-28509-z

BCG- disA -OE elicits greater macrophage reprogramming, phagocytic activity, and autophagy than BCG-WT in human and murine macrophages. a Percentages of inflammatory M1- and immunosuppressive M2-macrophages and M-MDSCs arising from primary murine macrophages. b Percentages of inflammatory, TNF-α + M1, and IL-6 + inflammatory macrophages (M1-like), and c CD206 + CD124 + and IL-10 + immunosuppressive macrophages (M2-like) arising from primary human macrophages. Data were collected after 24 h exposures at MOI of 20:1 as determined by flow-cytometry using gating schemes shown in Fig. S3 – S9 . Data are presented as mean values ± S.E.M. ( n = 4 independent biological replicate experiments). d Phagocytic activity in human primary macrophages in representative confocal photomicrographs showing intracellular uptake of FITC-labeled IgG-opsonized latex beads (green) with nuclei stained blue. Data are represented as mean values ± S.E.M ( n = 3 independent biological replicate experiments). e Autophagy induction and f quantification by BCG-LC3B colocalization in primary murine macrophages shown by representative confocal photomicrographs. Autophagy was measured by LC3B puncta or g . p62 colocalization with BCG appearing in yellow. FITC-labeled BCG strains are stained green, LC3B or p62 autophagic puncta (red), and nuclei blue. h Quantification of BCG-p62 colocalization. Cells were fixed using 4% paraformaldehyde 6 h after infection (MOI 10:1), and images obtained with an LSM700 confocal microscope and Fiji software processing. Quantification was measured by mean fluorescence intensity. Data represented for all confocal microscopy studies (phagocytosis, LC3B-BCG colocalization and p62-BCG colocalization) as mean values ± S.E.M. ( n = 3 independent biological replicate experiments). All statistical analyses done using two-tailed Student’s t -test (* P
Figure Legend Snippet: BCG- disA -OE elicits greater macrophage reprogramming, phagocytic activity, and autophagy than BCG-WT in human and murine macrophages. a Percentages of inflammatory M1- and immunosuppressive M2-macrophages and M-MDSCs arising from primary murine macrophages. b Percentages of inflammatory, TNF-α + M1, and IL-6 + inflammatory macrophages (M1-like), and c CD206 + CD124 + and IL-10 + immunosuppressive macrophages (M2-like) arising from primary human macrophages. Data were collected after 24 h exposures at MOI of 20:1 as determined by flow-cytometry using gating schemes shown in Fig. S3 – S9 . Data are presented as mean values ± S.E.M. ( n = 4 independent biological replicate experiments). d Phagocytic activity in human primary macrophages in representative confocal photomicrographs showing intracellular uptake of FITC-labeled IgG-opsonized latex beads (green) with nuclei stained blue. Data are represented as mean values ± S.E.M ( n = 3 independent biological replicate experiments). e Autophagy induction and f quantification by BCG-LC3B colocalization in primary murine macrophages shown by representative confocal photomicrographs. Autophagy was measured by LC3B puncta or g . p62 colocalization with BCG appearing in yellow. FITC-labeled BCG strains are stained green, LC3B or p62 autophagic puncta (red), and nuclei blue. h Quantification of BCG-p62 colocalization. Cells were fixed using 4% paraformaldehyde 6 h after infection (MOI 10:1), and images obtained with an LSM700 confocal microscope and Fiji software processing. Quantification was measured by mean fluorescence intensity. Data represented for all confocal microscopy studies (phagocytosis, LC3B-BCG colocalization and p62-BCG colocalization) as mean values ± S.E.M. ( n = 3 independent biological replicate experiments). All statistical analyses done using two-tailed Student’s t -test (* P

Techniques Used: Activity Assay, Flow Cytometry, Labeling, Staining, Infection, Microscopy, Software, Fluorescence, Confocal Microscopy, Two Tailed Test

17) Product Images from "Autism-associated chromatin remodeler CHD8 regulates erythroblast cytokinesis and fine-tunes the balance of Rho GTPase signaling"

Article Title: Autism-associated chromatin remodeler CHD8 regulates erythroblast cytokinesis and fine-tunes the balance of Rho GTPase signaling

Journal: Cell reports

doi: 10.1016/j.celrep.2022.111072

Defective Rho GTPase signaling is involved in cytokinesis failure in Chd8 −/− P53 −/− erythroblasts (A) Location of CHD8 binding peaks in sorted CD44 + TER119 + cells identified by CHD8 CUT RUN assay. (B) Genes overlapping between CHD8 binding targets and differentially expressed genes (DEGs) identified in Chd8 −/− P53 −/− cells through RNA-seq. (C) Reactome gene set enrichment with the differentially expressed CHD8 direct targets by Metascape website. (D) Representative CHD8 Cut Run tracks of Rhof , Arfgap3 , Arhgap11a , Pak6 , Lbr , Limk1 , and Arpc2 in WT erythroblasts generated by IGV software. (E) Expression heatmap of CHD8 target genes enriched in signaling by Rho GTPases in RNA-seq data. (F) RhoA-GTP level detected by pull-down assays with Rhotekin beads and western blotting (WB) of pMLC2 Ser19 and MLC2 in CD45 − TER119 + BM cells of WT, P53 −/− , and Chd8 −/− P53 −/− mice. (G) Rac1-GTP and Cdc42-GTP levels in each genotype detected by pull-down assays with PAK beads in CD45 − TER119 + BM cells. In (F) and (G), the number indicates the mean intensity level derived from 2 independent experiments. Protein levels were normalized to β-actin with levels in WT cells arbitrarily set at 1. Each experiment includes 3 mice for each genotype, and TER119 + cells were pulled together and used for protein extraction.
Figure Legend Snippet: Defective Rho GTPase signaling is involved in cytokinesis failure in Chd8 −/− P53 −/− erythroblasts (A) Location of CHD8 binding peaks in sorted CD44 + TER119 + cells identified by CHD8 CUT RUN assay. (B) Genes overlapping between CHD8 binding targets and differentially expressed genes (DEGs) identified in Chd8 −/− P53 −/− cells through RNA-seq. (C) Reactome gene set enrichment with the differentially expressed CHD8 direct targets by Metascape website. (D) Representative CHD8 Cut Run tracks of Rhof , Arfgap3 , Arhgap11a , Pak6 , Lbr , Limk1 , and Arpc2 in WT erythroblasts generated by IGV software. (E) Expression heatmap of CHD8 target genes enriched in signaling by Rho GTPases in RNA-seq data. (F) RhoA-GTP level detected by pull-down assays with Rhotekin beads and western blotting (WB) of pMLC2 Ser19 and MLC2 in CD45 − TER119 + BM cells of WT, P53 −/− , and Chd8 −/− P53 −/− mice. (G) Rac1-GTP and Cdc42-GTP levels in each genotype detected by pull-down assays with PAK beads in CD45 − TER119 + BM cells. In (F) and (G), the number indicates the mean intensity level derived from 2 independent experiments. Protein levels were normalized to β-actin with levels in WT cells arbitrarily set at 1. Each experiment includes 3 mice for each genotype, and TER119 + cells were pulled together and used for protein extraction.

Techniques Used: Binding Assay, RNA Sequencing Assay, Generated, Software, Expressing, Western Blot, Mouse Assay, Derivative Assay, Protein Extraction

18) Product Images from "Immune recognition of syngeneic, allogeneic and xenogeneic stromal cell transplants in healthy retinas"

Article Title: Immune recognition of syngeneic, allogeneic and xenogeneic stromal cell transplants in healthy retinas

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-022-03129-y

Graphical summary of the effects of BM-MSC transplantation type on the healthy retina. Syngeneic transplants induce fewer changes in the retina than allotransplants or allotransplant with immunosuppression (orange) and these than xenotransplants. Recruitment of CD45 + cells and microglial and Müller cell activation refer to the whole retina
Figure Legend Snippet: Graphical summary of the effects of BM-MSC transplantation type on the healthy retina. Syngeneic transplants induce fewer changes in the retina than allotransplants or allotransplant with immunosuppression (orange) and these than xenotransplants. Recruitment of CD45 + cells and microglial and Müller cell activation refer to the whole retina

Techniques Used: Transplantation Assay, Activation Assay

BM-MSC transplants trigger Müller cell hypertrophy, microglial activation and recruitment of CD45 + cells. A: Retinal magnifications from intact pigmented and albino animals showing the normal expression of vimentin in Müller cells (purple). B: Vimentin expression (purple) in transplanted retinas at 5 and 21 days. C : Column graph showing the signal intensity of vimentin relative to intact (mean signal in albino and pigmented, 100% arbitrary units) in all transplanted retinas at both time points ( n = 4 retinas/group/timepoint, 3 sections/retina). Müller cell hypertrophy was significant in allotransplanted retinas at 5 and 21 days and in xenotransplanted retinas at 21 days (* p
Figure Legend Snippet: BM-MSC transplants trigger Müller cell hypertrophy, microglial activation and recruitment of CD45 + cells. A: Retinal magnifications from intact pigmented and albino animals showing the normal expression of vimentin in Müller cells (purple). B: Vimentin expression (purple) in transplanted retinas at 5 and 21 days. C : Column graph showing the signal intensity of vimentin relative to intact (mean signal in albino and pigmented, 100% arbitrary units) in all transplanted retinas at both time points ( n = 4 retinas/group/timepoint, 3 sections/retina). Müller cell hypertrophy was significant in allotransplanted retinas at 5 and 21 days and in xenotransplanted retinas at 21 days (* p

Techniques Used: Activation Assay, Expressing

19) Product Images from "Zinc-modified titanium surface enhances osteoblast differentiation of dental pulp stem cells in vitro"

Article Title: Zinc-modified titanium surface enhances osteoblast differentiation of dental pulp stem cells in vitro

Journal: Scientific Reports

doi: 10.1038/srep29462

Characterization and flow cytometric analysis of DPSCs. DPSCs were positive for MSC markers CD44 and CD73 and were negative for hematopoietic markers CD45 and CD14.
Figure Legend Snippet: Characterization and flow cytometric analysis of DPSCs. DPSCs were positive for MSC markers CD44 and CD73 and were negative for hematopoietic markers CD45 and CD14.

Techniques Used: Flow Cytometry

20) Product Images from "A Soluble Granulocyte Colony Stimulating Factor Decoy Receptor as a Novel Tool to Increase Hematopoietic Cell Homing and Reconstitution in Mice"

Article Title: A Soluble Granulocyte Colony Stimulating Factor Decoy Receptor as a Novel Tool to Increase Hematopoietic Cell Homing and Reconstitution in Mice

Journal: Stem Cells and Development

doi: 10.1089/scd.2012.0438

Altered homing and engraftment by the secretion of a soluble G-CSF decoy receptor. (A) Schematic of the experiment. In brief, CD45.2 mice were sublethally irradiated at the dose of 8 Gy and immediately injected intraperitoneally (i.p.) with PBS alone
Figure Legend Snippet: Altered homing and engraftment by the secretion of a soluble G-CSF decoy receptor. (A) Schematic of the experiment. In brief, CD45.2 mice were sublethally irradiated at the dose of 8 Gy and immediately injected intraperitoneally (i.p.) with PBS alone

Techniques Used: Mouse Assay, Irradiation, Injection

21) Product Images from "Chronic social stress induces peripheral and central immune activation, blunted mesolimbic dopamine function, and reduced reward-directed behaviour in mice"

Article Title: Chronic social stress induces peripheral and central immune activation, blunted mesolimbic dopamine function, and reduced reward-directed behaviour in mice

Journal: Neurobiology of Stress

doi: 10.1016/j.ynstr.2018.01.004

Effects of CSS on splenic leukocytes at day 16 assessed using flow cytometry (Expt 1). Spleens were processed from 6 CSS and 6 CON mice. A) FACS dot plots for gating of lymphocytes and myeloid cells based on CD45 and CD11b staining, in representative CON and CSS mice. B) Cell counts for lymphocytes (CD45 + /CD11b − ). C) Cell counts for myeloid cells (CD45 + /CD11b + ). D) FACS dot plots for total myeloid cells gated based on SSC and Ly6C staining, in representative CON and CSS mice. E) Cell counts for granulocytes (SSC hi /Ly6C int ). F) Cell counts for inflammatory monocytes (SSC lo /Ly6C hi ). G - I) Expression levels of activation markers in splenic myeloid cells (CD45 + /CD11b + ); marker expression is measured as the geometric mean of fluorescence intensity (MFI). G) MHC II, H) TNF-α, I) IFN-γ. In H) and I) the data point for one CSS mouse was excluded because of a methodological problem with the intracellular staining. p values were obtained in unpaired t -tests. CD45, cluster of differentiation 45; CD11b, cluster of differentiation 11b; SSC, side scatter of cells; Ly6C, lymphocyte antigen 6C.
Figure Legend Snippet: Effects of CSS on splenic leukocytes at day 16 assessed using flow cytometry (Expt 1). Spleens were processed from 6 CSS and 6 CON mice. A) FACS dot plots for gating of lymphocytes and myeloid cells based on CD45 and CD11b staining, in representative CON and CSS mice. B) Cell counts for lymphocytes (CD45 + /CD11b − ). C) Cell counts for myeloid cells (CD45 + /CD11b + ). D) FACS dot plots for total myeloid cells gated based on SSC and Ly6C staining, in representative CON and CSS mice. E) Cell counts for granulocytes (SSC hi /Ly6C int ). F) Cell counts for inflammatory monocytes (SSC lo /Ly6C hi ). G - I) Expression levels of activation markers in splenic myeloid cells (CD45 + /CD11b + ); marker expression is measured as the geometric mean of fluorescence intensity (MFI). G) MHC II, H) TNF-α, I) IFN-γ. In H) and I) the data point for one CSS mouse was excluded because of a methodological problem with the intracellular staining. p values were obtained in unpaired t -tests. CD45, cluster of differentiation 45; CD11b, cluster of differentiation 11b; SSC, side scatter of cells; Ly6C, lymphocyte antigen 6C.

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, FACS, Staining, Expressing, Activation Assay, Marker, Fluorescence

Effects of CSS on brain measures of immune status at day 16 (Expts 1, 3 and 4). Expt 1: Fresh brains were obtained from 6 CSS and 6 CON mice (same mice as in Fig. 1 , Fig. 2 ). A) FACS dot plots from representative CON and CSS mice for mononuclear cells from whole brain tissue showing gating of lymphocytes (CD11b − /CD45 + ) and CD11b + cells (left-hand dot plots); within CD11b + cells, CD45 staining was used to gate microglia (CD45 int ) and brain-infiltrating myeloid cells (CD45 hi ) (right-hand dot plots). B) Cell counts for microglia. C) Cell counts for myeloid cells. Expt 3: Perfused brains were obtained from 7 CSS and 8 CON mice were sectioned and immunostained for the microglia marker Iba1. D) Representative images of Iba1 staining in the ventral tegmental area (VTA, demarcated with dashed white lines) from a CON mouse and a CSS mouse. E) Percentage Iba1 + immunoreactive (IR) area in the VTA. Expt 4: Fresh fixed brains were obtained from 12 CSS and 10 CON mice. F) Comparison of VTA expression of CD11b mRNA measured using qPCR. p values were obtained in unpaired t -tests.
Figure Legend Snippet: Effects of CSS on brain measures of immune status at day 16 (Expts 1, 3 and 4). Expt 1: Fresh brains were obtained from 6 CSS and 6 CON mice (same mice as in Fig. 1 , Fig. 2 ). A) FACS dot plots from representative CON and CSS mice for mononuclear cells from whole brain tissue showing gating of lymphocytes (CD11b − /CD45 + ) and CD11b + cells (left-hand dot plots); within CD11b + cells, CD45 staining was used to gate microglia (CD45 int ) and brain-infiltrating myeloid cells (CD45 hi ) (right-hand dot plots). B) Cell counts for microglia. C) Cell counts for myeloid cells. Expt 3: Perfused brains were obtained from 7 CSS and 8 CON mice were sectioned and immunostained for the microglia marker Iba1. D) Representative images of Iba1 staining in the ventral tegmental area (VTA, demarcated with dashed white lines) from a CON mouse and a CSS mouse. E) Percentage Iba1 + immunoreactive (IR) area in the VTA. Expt 4: Fresh fixed brains were obtained from 12 CSS and 10 CON mice. F) Comparison of VTA expression of CD11b mRNA measured using qPCR. p values were obtained in unpaired t -tests.

Techniques Used: Mouse Assay, FACS, Staining, Marker, Expressing, Real-time Polymerase Chain Reaction

22) Product Images from "Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage"

Article Title: Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.11051

Expression of markers in human AFSCs. ( A ) Expression of surface antigens was carried out by flow cytometry using mouse monoclonal antibodies (filled curve). All panels include an isotype-matched negative control (unfilled curve). Antigens tested were, as indicated on panels: CD73, CD90, CD105, CD29, CD34, CD133, CD106, CD45, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. ( B ) RT-PCR was performed on five human AFSC cell lines and a human ESC cell line (positive control), using primers against OCT-4, SOX2, NANOG, KLF4, C-MYC, REX1, and LIN28 .
Figure Legend Snippet: Expression of markers in human AFSCs. ( A ) Expression of surface antigens was carried out by flow cytometry using mouse monoclonal antibodies (filled curve). All panels include an isotype-matched negative control (unfilled curve). Antigens tested were, as indicated on panels: CD73, CD90, CD105, CD29, CD34, CD133, CD106, CD45, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. ( B ) RT-PCR was performed on five human AFSC cell lines and a human ESC cell line (positive control), using primers against OCT-4, SOX2, NANOG, KLF4, C-MYC, REX1, and LIN28 .

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Reverse Transcription Polymerase Chain Reaction, Positive Control

23) Product Images from "Interleukin-17A Enhances Host Defense against Cryptococcal Lung Infection through Effects Mediated by Leukocyte Recruitment, Activation, and Gamma Interferon Production"

Article Title: Interleukin-17A Enhances Host Defense against Cryptococcal Lung Infection through Effects Mediated by Leukocyte Recruitment, Activation, and Gamma Interferon Production

Journal: Infection and Immunity

doi: 10.1128/IAI.01477-13

Accumulations of total lung leukocytes, including the CD11c + CD11b + myeloid cell subset, are modestly impaired in IL-17A-deficient mice with persistent cryptococcal lung infection. (A, B) WT C57BL/6 mice and IL-17A −/− mice were infected by the intratracheal route with C. neoformans strain 52D. At weeks 0 (uninfected), 1, 2, 4, and 8, lungs were removed and enzymatically digested, and lung cells were antibody stained and analyzed by flow cytometric analysis to identify total leukocytes and leukocyte subsets as described in Materials and Methods. (A) Total CD45 + lung leukocytes. (B) Total numbers of neutrophils, eosinophils, CD11c + CD11b + myeloid cells (which include CD11b + dendritic cells and exudate macrophages), and CD11b − macrophages (which include resident alveolar macrophages). Black bars, WT mice; white bars, IL-17A −/− mice. Data are means ± standard errors obtained from 4 to 6 mice per time point in two separate experiments. *, P
Figure Legend Snippet: Accumulations of total lung leukocytes, including the CD11c + CD11b + myeloid cell subset, are modestly impaired in IL-17A-deficient mice with persistent cryptococcal lung infection. (A, B) WT C57BL/6 mice and IL-17A −/− mice were infected by the intratracheal route with C. neoformans strain 52D. At weeks 0 (uninfected), 1, 2, 4, and 8, lungs were removed and enzymatically digested, and lung cells were antibody stained and analyzed by flow cytometric analysis to identify total leukocytes and leukocyte subsets as described in Materials and Methods. (A) Total CD45 + lung leukocytes. (B) Total numbers of neutrophils, eosinophils, CD11c + CD11b + myeloid cells (which include CD11b + dendritic cells and exudate macrophages), and CD11b − macrophages (which include resident alveolar macrophages). Black bars, WT mice; white bars, IL-17A −/− mice. Data are means ± standard errors obtained from 4 to 6 mice per time point in two separate experiments. *, P

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

24) Product Images from "Dietary NaCl affects bleomycin-induced lung fibrosis in mice"

Article Title: Dietary NaCl affects bleomycin-induced lung fibrosis in mice

Journal: Experimental lung research

doi: 10.1080/01902148.2017.1385110

Dietary salt modulates bleomycin-induced CD45 positive cell accumulation in lungs Mice received oropharyngeal bleomycin or saline on day 0. Mice were maintained on low, regular, or high NaCl diets for 21 days, and lung sections were stained for the leukocyte marker CD45. Bar is 100 μ m. Images are representative of three independent experiments.
Figure Legend Snippet: Dietary salt modulates bleomycin-induced CD45 positive cell accumulation in lungs Mice received oropharyngeal bleomycin or saline on day 0. Mice were maintained on low, regular, or high NaCl diets for 21 days, and lung sections were stained for the leukocyte marker CD45. Bar is 100 μ m. Images are representative of three independent experiments.

Techniques Used: Mouse Assay, Staining, Marker

Dietary salt modulates bleomycin-induced fibrocyte accumulation in lungs Mice received oropharyngeal A–D) bleomycin or F–H) saline on day 0. Mice were maintained on A and F) low, B and G) regular, or C and H) high NaCl diets for 21 days. Lung sections were stained for CD45 (red) and collagen-VI (green) to detect fibrocytes (orange). Nuclei were counterstained with DAPI (blue). D) Sections were stained with control antibodies. Bar is 100 μm. Images are representative of three independent experiments. E) Quantification of fibrocytes. Values are mean ± SEM, n = 3–4 mice per group. * p
Figure Legend Snippet: Dietary salt modulates bleomycin-induced fibrocyte accumulation in lungs Mice received oropharyngeal A–D) bleomycin or F–H) saline on day 0. Mice were maintained on A and F) low, B and G) regular, or C and H) high NaCl diets for 21 days. Lung sections were stained for CD45 (red) and collagen-VI (green) to detect fibrocytes (orange). Nuclei were counterstained with DAPI (blue). D) Sections were stained with control antibodies. Bar is 100 μm. Images are representative of three independent experiments. E) Quantification of fibrocytes. Values are mean ± SEM, n = 3–4 mice per group. * p

Techniques Used: Mouse Assay, Staining

Dietary salt modulates bleomycin-induced leukocyte accumulation in lungs Mice received oropharyngeal bleomycin or saline on day 0. Mice were maintained on low, regular, or high NaCl diets for 21 days. A) Lung sections were stained for CD45 to detect all leukocytes, B) CD3 to detect T lymphocytes, C) CD11c to detect resident alveolar macrophages and dendritic cells, D) CD11b to detect blood and inflammatory macrophages, E) Ly6c to detect inflammatory macrophages, or F) CD206 to detect macrophages and dendritic cells. Values are mean ± SEM, n = 3–4 per group. * p
Figure Legend Snippet: Dietary salt modulates bleomycin-induced leukocyte accumulation in lungs Mice received oropharyngeal bleomycin or saline on day 0. Mice were maintained on low, regular, or high NaCl diets for 21 days. A) Lung sections were stained for CD45 to detect all leukocytes, B) CD3 to detect T lymphocytes, C) CD11c to detect resident alveolar macrophages and dendritic cells, D) CD11b to detect blood and inflammatory macrophages, E) Ly6c to detect inflammatory macrophages, or F) CD206 to detect macrophages and dendritic cells. Values are mean ± SEM, n = 3–4 per group. * p

Techniques Used: Mouse Assay, Staining

25) Product Images from "Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age"

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

Journal: Nature metabolism

doi: 10.1038/s42255-020-0228-3

Transfer of young eosinophils is associated with alterations in muscle stem cell frequencies but not function. (a) Gating strategy and representative flow plots of CD31 – , CD45 – , Sca-1 – , Vcam + and integrin α7 + satellite cells in muscle of Young ( n =13), Aged-PBS ( n =17), Aged-yEOS, ( n =17) and Aged-yEOS IL−4−/− ( n =17) mice. (b) Quantification of muscle stem cell frequencies in indicated groups (c) Representative photographs of immunofluorescent stained sort-purified and differentiated satellite cells. (d) Quantification of cell colony formation of sort-purified muscle stem cells of Young ( n =10), Aged-PBS ( n =10), Aged-yEOS ( n =8) and Aged-yEOS IL−4−/− ( n =8) mice (e) Representative H E stained longitudinal and cross-sectional quadriceps femoris in indicated groups. (f) Quantification of centrally nucleated myofibers in sections of Young ( n =26), Aged-PBS ( n =26), Aged-yEOS ( n =18) and Aged-yEOS IL−4−/− ( n =13) mice. (g) Muscle weight (femur) was measured in Young ( n =5), Aged-PBS ( n =9), Aged-yEOS ( n =8) and Aged-yEOS IL−4−/− ( n =7) mice. Data (a-f) are pooled from 2 independently performed experiments except for g (one experiment has been performed). Statistical significance was calculated by one-way ANOVA followed by two-tailed post-hoc Dunnett’s multiple comparison test against the aged-PBS treated group. Data are shown as individual data points with mean ± SEM. *p
Figure Legend Snippet: Transfer of young eosinophils is associated with alterations in muscle stem cell frequencies but not function. (a) Gating strategy and representative flow plots of CD31 – , CD45 – , Sca-1 – , Vcam + and integrin α7 + satellite cells in muscle of Young ( n =13), Aged-PBS ( n =17), Aged-yEOS, ( n =17) and Aged-yEOS IL−4−/− ( n =17) mice. (b) Quantification of muscle stem cell frequencies in indicated groups (c) Representative photographs of immunofluorescent stained sort-purified and differentiated satellite cells. (d) Quantification of cell colony formation of sort-purified muscle stem cells of Young ( n =10), Aged-PBS ( n =10), Aged-yEOS ( n =8) and Aged-yEOS IL−4−/− ( n =8) mice (e) Representative H E stained longitudinal and cross-sectional quadriceps femoris in indicated groups. (f) Quantification of centrally nucleated myofibers in sections of Young ( n =26), Aged-PBS ( n =26), Aged-yEOS ( n =18) and Aged-yEOS IL−4−/− ( n =13) mice. (g) Muscle weight (femur) was measured in Young ( n =5), Aged-PBS ( n =9), Aged-yEOS ( n =8) and Aged-yEOS IL−4−/− ( n =7) mice. Data (a-f) are pooled from 2 independently performed experiments except for g (one experiment has been performed). Statistical significance was calculated by one-way ANOVA followed by two-tailed post-hoc Dunnett’s multiple comparison test against the aged-PBS treated group. Data are shown as individual data points with mean ± SEM. *p

Techniques Used: Mouse Assay, Staining, Purification, Two Tailed Test

26) Product Images from "Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma"

Article Title: Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma

Journal: Scientific Reports

doi: 10.1038/srep25220

Trypan blue or DAPI exclusion overestimates tumour cell viability which is heterogeneous in tumoural subpopulations. ( A ) Trypan blue exclusion and DAPI exclusion give equivalent estimates of cell viability in matched and unmatched fresh primary patient samples. ( B ) In freshly processed ex vivo ccRCC patient samples, cell viability is overestimated by DAPI exclusion alone, as a high proportion of DAPI-negative cells are Annexin-V positive (green box). Truly viable Annexin-V − /DAPI − cells are in the left lower quadrant (orange box). ( C ) Comparison of viability assessment in CD45 + and CD45 neg fractions using DAPI alone (blue) or DAPI and Annexin-V (orange). DAPI alone significantly overestimates viability, by ~4.7X (p = 0.0002) in the CD45 neg compartment. Each point represents an individual patient. ( D ) A deeper examination of viability in different tumour cell sub-compartments reveals that while CD45 + cells and fibroblasts have reasonable viability, endothelial cells were uniformly apoptotic or dead. Tumour cell viability remained very low in these experiments. Data in C and D are represented as mean ± SEM. See Supplementary Figure 6 for gating strategy.
Figure Legend Snippet: Trypan blue or DAPI exclusion overestimates tumour cell viability which is heterogeneous in tumoural subpopulations. ( A ) Trypan blue exclusion and DAPI exclusion give equivalent estimates of cell viability in matched and unmatched fresh primary patient samples. ( B ) In freshly processed ex vivo ccRCC patient samples, cell viability is overestimated by DAPI exclusion alone, as a high proportion of DAPI-negative cells are Annexin-V positive (green box). Truly viable Annexin-V − /DAPI − cells are in the left lower quadrant (orange box). ( C ) Comparison of viability assessment in CD45 + and CD45 neg fractions using DAPI alone (blue) or DAPI and Annexin-V (orange). DAPI alone significantly overestimates viability, by ~4.7X (p = 0.0002) in the CD45 neg compartment. Each point represents an individual patient. ( D ) A deeper examination of viability in different tumour cell sub-compartments reveals that while CD45 + cells and fibroblasts have reasonable viability, endothelial cells were uniformly apoptotic or dead. Tumour cell viability remained very low in these experiments. Data in C and D are represented as mean ± SEM. See Supplementary Figure 6 for gating strategy.

Techniques Used: Ex Vivo

27) Product Images from "Tumor-Promoting Ly-6G+ SiglecFhigh Cells Are Mature and Long-Lived Neutrophils"

Article Title: Tumor-Promoting Ly-6G+ SiglecFhigh Cells Are Mature and Long-Lived Neutrophils

Journal: Cell Reports

doi: 10.1016/j.celrep.2020.108164

SiglecF high Neutrophils Are Long-Lived Cells within Tumors (A) Representative Ly-6G (top) and SiglecF (bottom) staining on cryopreserved KP1.9 lung tumor tissue sections of mice euthanized on day 29 after intravenous tumor cell injection. Tumor areas are highlighted by dotted pink lines. Scale bar, 25 μm. (B) Diagram describing parabiosis and separation procedures on KP1.9 tumor-bearing mice (CD45.1 and CD45.2) to investigate the lifespan of parabiont-derived SiglecF high and SiglecF low neutrophils in blood and lung tumors at different time points after separation (days 0, 1, 4, and 6). Mice were parabiosed 1 week after intravenous KP1.9 tumor-cell injection and separated on day 29. (C) Lung weight of parabiosed tumor-bearing mice (n = 5–6 mice/time point) at time of euthanasia following separation. The lung weight of non-parabiosed tumor-free mice is shown as control (n = 4 mice). (D) Quantification by flow cytometry of parabiont-derived CD11b + Ly-6G + neutrophils in blood of tumor-bearing mice (n = 5–6 mice/time point) at indicated post-separation time points. (E) Flow-cytometry-based quantification of lung tumor-infiltrating SiglecF low (left) and SiglecF high neutrophils (right) derived from the respective parabiont (n = 5–6 mice/time point). (F) Percent of parabiont-derived neutrophils in blood and tumor-bearing lung after separation (n = 5–6 mice/time point). The dashed line indicates the half-life of SiglecF high neutrophils in tumor-bearing lung tissue. Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between three or more groups, one-way ANOVA with multiple comparisons was used. ∗ p
Figure Legend Snippet: SiglecF high Neutrophils Are Long-Lived Cells within Tumors (A) Representative Ly-6G (top) and SiglecF (bottom) staining on cryopreserved KP1.9 lung tumor tissue sections of mice euthanized on day 29 after intravenous tumor cell injection. Tumor areas are highlighted by dotted pink lines. Scale bar, 25 μm. (B) Diagram describing parabiosis and separation procedures on KP1.9 tumor-bearing mice (CD45.1 and CD45.2) to investigate the lifespan of parabiont-derived SiglecF high and SiglecF low neutrophils in blood and lung tumors at different time points after separation (days 0, 1, 4, and 6). Mice were parabiosed 1 week after intravenous KP1.9 tumor-cell injection and separated on day 29. (C) Lung weight of parabiosed tumor-bearing mice (n = 5–6 mice/time point) at time of euthanasia following separation. The lung weight of non-parabiosed tumor-free mice is shown as control (n = 4 mice). (D) Quantification by flow cytometry of parabiont-derived CD11b + Ly-6G + neutrophils in blood of tumor-bearing mice (n = 5–6 mice/time point) at indicated post-separation time points. (E) Flow-cytometry-based quantification of lung tumor-infiltrating SiglecF low (left) and SiglecF high neutrophils (right) derived from the respective parabiont (n = 5–6 mice/time point). (F) Percent of parabiont-derived neutrophils in blood and tumor-bearing lung after separation (n = 5–6 mice/time point). The dashed line indicates the half-life of SiglecF high neutrophils in tumor-bearing lung tissue. Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between three or more groups, one-way ANOVA with multiple comparisons was used. ∗ p

Techniques Used: Staining, Mouse Assay, Injection, Derivative Assay, Flow Cytometry, Two Tailed Test

SiglecF high CD11b + Ly-6G + Cells Resemble Neutrophils (A) Cells obtained from lung tissue of KP1.9 tumor-bearing mice (day 29 after intravenous tumor cell injection) were stained by flow cytometry to identify eosinophils (CD11b + Ly-6G − SiglecF + ), SiglecF low (CD11b + Ly-6G + SiglecF low ), and SiglecF high neutrophils (CD11b + Ly-6G + SiglecF high ). Representative dot plots are shown (pre-gated on live cells). (B) Suspensions of CD45 + cells for single-cell RNA sequencing were prepared from murine KP1.9 lung tumors (T) (n = 2) and lung tissue of healthy mice (H) (n = 2; Engblom et al., 2017 ; Zilionis et al., 2019 ). Major cell types (neutrophils highlighted in red) were identified by a Bayesian cell classifier as reported in Zilionis et al. (2019) , and neutrophils were defined as tumor (T)- Siglecf high , tumor (T)- Siglecf low , and healthy (H)- Siglecf low based on the expression of genes correlated to SiglecF ( Engblom et al., 2017 ). The heatmap shows a comparison of the 3 neutrophil subsets and of alveolar macrophages (Mø 4 ), monocytes, dendritic cells (DCs), and basophils (rows) to immune profiles defined by the Immgen consortium (columns). (C) Representative histogram (left) and quantification of geometric mean fluorescence intensity (gMFI) followed by fluorescence-minus one (FMO) signal subtraction (right) of CCR3 expression measured by flow cytometry in eosinophils and SiglecF low and SiglecF high neutrophils (day 29 after intravenous tumor cell injection; n = 4 mice/group). (D) Representative histogram (left) and quantification of delta gMFI (right) of SiglecE expression measured by flow cytometry in eosinophils and SiglecF low and SiglecF high neutrophils (day 29 after intravenous tumor cell injection; n = 4 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. ∗∗∗∗ p
Figure Legend Snippet: SiglecF high CD11b + Ly-6G + Cells Resemble Neutrophils (A) Cells obtained from lung tissue of KP1.9 tumor-bearing mice (day 29 after intravenous tumor cell injection) were stained by flow cytometry to identify eosinophils (CD11b + Ly-6G − SiglecF + ), SiglecF low (CD11b + Ly-6G + SiglecF low ), and SiglecF high neutrophils (CD11b + Ly-6G + SiglecF high ). Representative dot plots are shown (pre-gated on live cells). (B) Suspensions of CD45 + cells for single-cell RNA sequencing were prepared from murine KP1.9 lung tumors (T) (n = 2) and lung tissue of healthy mice (H) (n = 2; Engblom et al., 2017 ; Zilionis et al., 2019 ). Major cell types (neutrophils highlighted in red) were identified by a Bayesian cell classifier as reported in Zilionis et al. (2019) , and neutrophils were defined as tumor (T)- Siglecf high , tumor (T)- Siglecf low , and healthy (H)- Siglecf low based on the expression of genes correlated to SiglecF ( Engblom et al., 2017 ). The heatmap shows a comparison of the 3 neutrophil subsets and of alveolar macrophages (Mø 4 ), monocytes, dendritic cells (DCs), and basophils (rows) to immune profiles defined by the Immgen consortium (columns). (C) Representative histogram (left) and quantification of geometric mean fluorescence intensity (gMFI) followed by fluorescence-minus one (FMO) signal subtraction (right) of CCR3 expression measured by flow cytometry in eosinophils and SiglecF low and SiglecF high neutrophils (day 29 after intravenous tumor cell injection; n = 4 mice/group). (D) Representative histogram (left) and quantification of delta gMFI (right) of SiglecE expression measured by flow cytometry in eosinophils and SiglecF low and SiglecF high neutrophils (day 29 after intravenous tumor cell injection; n = 4 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. ∗∗∗∗ p

Techniques Used: Mouse Assay, Injection, Staining, Flow Cytometry, RNA Sequencing Assay, Expressing, Fluorescence, Two Tailed Test

SiglecF high Neutrophils Continuously Accumulate in Tumor-Bearing Lungs (A) Diagram describing experimental procedure of the analysis of lung cells obtained from tumor-free or tumor-bearing mice. (B) Lung weight (n = 5–14 mice/group) of tumor-free and KP1.9 tumor-bearing mice (days 5, 19, or 32 after intravenous tumor cell injection). (C) Flow-cytometry-based detection of SiglecF high and SiglecF low neutrophils from healthy lung tissue and KP1.9 lung tumors at different time points after tumor cell injection. Representative dot plots are shown (pre-gated on live CD45 + Lineage [Lin] − CD11b + cells). The lineage master mix contained antibodies specific for CD90.2 and B220. (D) Quantification of SiglecF low (left) and SiglecF high (right) neutrophil numbers per mg lung tissue in tumor-free and tumor-bearing mice detected by flow cytometry (n = 5–14 mice/group). (E) Lung SiglecF low (left) and SiglecF high (right) neutrophils (percent of live cells measured by flow cytometry) plotted against lung weight (proxy of tumor burden) of KP1.9 lung-tumor-bearing or tumor-free mice (n = 34 mice) and linear regression was performed. (F) Outline of experimental procedure for analysis of bronchoalveolar lavage (BAL) fluid-derived cells from tumor-free and KP1.9 lung cancer-bearing mice. (G) Flow-cytometry-based detection of SiglecF high and SiglecF low neutrophils from BAL fluid of healthy mice and mice carrying KP1.9 lung tumors at different time points after tumor cell injection. Representative dot plots are shown (pre-gated on live CD45 + Lin − CD11b + Ly-6G + cells). The lineage master mix contained antibodies specific for CD90.2 and B220. (H) Quantification of SiglecF low (top) and SiglecF high (bottom) neutrophils in BAL fluid of tumor-free and tumor-bearing mice detected by flow cytometry (n = 5–13 mice/group). (I) Neutrophils in BAL fluid of KP1.9 lung-tumor-bearing or tumor-free mice (n = 30 mice) plotted against neutrophils in lung tissue, with performed linear regression. Data for SiglecF low (left) and SiglecF high (right) neutrophils were measured by flow cytometry, and percent of live cells is shown. (J) BAL fluid SiglecF low (left) and SiglecF high (right) neutrophils (percent of live cells measured by flow cytometry) plotted against lung weight of KP1.9 lung-tumor-bearing or tumor-free mice (n = 30 mice) and linear regression was performed. Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. ∗ p
Figure Legend Snippet: SiglecF high Neutrophils Continuously Accumulate in Tumor-Bearing Lungs (A) Diagram describing experimental procedure of the analysis of lung cells obtained from tumor-free or tumor-bearing mice. (B) Lung weight (n = 5–14 mice/group) of tumor-free and KP1.9 tumor-bearing mice (days 5, 19, or 32 after intravenous tumor cell injection). (C) Flow-cytometry-based detection of SiglecF high and SiglecF low neutrophils from healthy lung tissue and KP1.9 lung tumors at different time points after tumor cell injection. Representative dot plots are shown (pre-gated on live CD45 + Lineage [Lin] − CD11b + cells). The lineage master mix contained antibodies specific for CD90.2 and B220. (D) Quantification of SiglecF low (left) and SiglecF high (right) neutrophil numbers per mg lung tissue in tumor-free and tumor-bearing mice detected by flow cytometry (n = 5–14 mice/group). (E) Lung SiglecF low (left) and SiglecF high (right) neutrophils (percent of live cells measured by flow cytometry) plotted against lung weight (proxy of tumor burden) of KP1.9 lung-tumor-bearing or tumor-free mice (n = 34 mice) and linear regression was performed. (F) Outline of experimental procedure for analysis of bronchoalveolar lavage (BAL) fluid-derived cells from tumor-free and KP1.9 lung cancer-bearing mice. (G) Flow-cytometry-based detection of SiglecF high and SiglecF low neutrophils from BAL fluid of healthy mice and mice carrying KP1.9 lung tumors at different time points after tumor cell injection. Representative dot plots are shown (pre-gated on live CD45 + Lin − CD11b + Ly-6G + cells). The lineage master mix contained antibodies specific for CD90.2 and B220. (H) Quantification of SiglecF low (top) and SiglecF high (bottom) neutrophils in BAL fluid of tumor-free and tumor-bearing mice detected by flow cytometry (n = 5–13 mice/group). (I) Neutrophils in BAL fluid of KP1.9 lung-tumor-bearing or tumor-free mice (n = 30 mice) plotted against neutrophils in lung tissue, with performed linear regression. Data for SiglecF low (left) and SiglecF high (right) neutrophils were measured by flow cytometry, and percent of live cells is shown. (J) BAL fluid SiglecF low (left) and SiglecF high (right) neutrophils (percent of live cells measured by flow cytometry) plotted against lung weight of KP1.9 lung-tumor-bearing or tumor-free mice (n = 30 mice) and linear regression was performed. Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. ∗ p

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Derivative Assay, Two Tailed Test

28) Product Images from "The Chemokine Receptor CXCR4 Mediates Recruitment of CD11c+ Conventional Dendritic Cells Into the Inflamed Murine Cornea"

Article Title: The Chemokine Receptor CXCR4 Mediates Recruitment of CD11c+ Conventional Dendritic Cells Into the Inflamed Murine Cornea

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.18-25084

CXCR4 chemokine receptor expression increase in the inflamed murine cornea. Representative confocal micrographs of 7-day suture inflamed BALB/c corneas show increased CD11c + cells (red, A, D), CXCR4 + cells (green, B, E), and colocalization (yellow, C, F) throughout the corneal stroma of the peripheral (A–C) and central corneas (D–F). Orthogonal views highlight distribution throughout each corneal area. Cell density quantification of CD11c + and CXCR4 + cells in peripheral and central corneal stroma (cells/mm 2 ) highlights populations of CXCR4 + cDCs to be present following inflammation: significance for cell densities between central and peripheral cornea is denoted by asterisks (G). Representative histograms of flow cytometry analysis of relative CXCR4 expression within CD45 + leukocytes in the inflamed corneal epithelium (H) and stroma (I). Data are shown as mean ± SEM. t-test, **P
Figure Legend Snippet: CXCR4 chemokine receptor expression increase in the inflamed murine cornea. Representative confocal micrographs of 7-day suture inflamed BALB/c corneas show increased CD11c + cells (red, A, D), CXCR4 + cells (green, B, E), and colocalization (yellow, C, F) throughout the corneal stroma of the peripheral (A–C) and central corneas (D–F). Orthogonal views highlight distribution throughout each corneal area. Cell density quantification of CD11c + and CXCR4 + cells in peripheral and central corneal stroma (cells/mm 2 ) highlights populations of CXCR4 + cDCs to be present following inflammation: significance for cell densities between central and peripheral cornea is denoted by asterisks (G). Representative histograms of flow cytometry analysis of relative CXCR4 expression within CD45 + leukocytes in the inflamed corneal epithelium (H) and stroma (I). Data are shown as mean ± SEM. t-test, **P

Techniques Used: Expressing, Flow Cytometry, Cytometry

CXCL12 expression in the naïve and inflamed cornea. Representative confocal micrograph of naïve (A) and inflamed (B) BALB/c corneas, with the respective isotype control (C), show expression of CD11c + cDCs (green) and CXCL12 (red) within the corneal stroma. The orthogonal views reveal CXCL12 intensity from the naïve and inflamed corneal epithelium. Further, the white dotted box denoted within corresponding orthogonal views below each en face image highlights the area presented in the top panel images. CXCL12 mRNA expression is upregulated in the cornea 7 and 14 days after suture placement compared with naïve corneas (D). Representative histograms of flow cytometric analysis of CXCL12 expression within CD45 + leukocytes in the naïve (E) and inflamed (F) corneal epithelium and stromal layers. Scale bars denote 50 μm. Data are shown as mean ± SEM. *P
Figure Legend Snippet: CXCL12 expression in the naïve and inflamed cornea. Representative confocal micrograph of naïve (A) and inflamed (B) BALB/c corneas, with the respective isotype control (C), show expression of CD11c + cDCs (green) and CXCL12 (red) within the corneal stroma. The orthogonal views reveal CXCL12 intensity from the naïve and inflamed corneal epithelium. Further, the white dotted box denoted within corresponding orthogonal views below each en face image highlights the area presented in the top panel images. CXCL12 mRNA expression is upregulated in the cornea 7 and 14 days after suture placement compared with naïve corneas (D). Representative histograms of flow cytometric analysis of CXCL12 expression within CD45 + leukocytes in the naïve (E) and inflamed (F) corneal epithelium and stromal layers. Scale bars denote 50 μm. Data are shown as mean ± SEM. *P

Techniques Used: Expressing, Flow Cytometry

CXCR4 chemokine receptor is constitutively expressed in the naïve murine cornea. Representative confocal micrographs of naïve BALB/c corneas in peripheral (A–C) and central (D–F) stroma show expression of CD11c + (red, A, D) and CXCR4 + (green, B, E) and colocalization (yellow, C, F) throughout the corneal stroma. Orthogonal views highlighting positive staining added to each corneal area. White dotted boxes show examples of CD11c + DCs (magnified in A–C, stroma (i) and epithelium (ii); and D–F insets), as well as a CD11c − cells (magnified in A–C, inset i) coexpressing CXCR4 in the peripheral and central corneas. Isotype controls for CD11c and CXCR4 do not show positive staining (G–I). Cell density quantification for CD11c + and CXCR4 + cells (cells/mm 2 ) highlights populations of steady-state cDCs expressing CXCR4: significance for cell densities between central and peripheral cornea is denoted by an asterisk (J). Representative histograms of flow cytometry analysis of relative CXCR4 expression of CD45 + leukocytes in the inflamed corneal epithelium (K) and stroma (L). Data are shown as mean ± SEM. t-test, *P
Figure Legend Snippet: CXCR4 chemokine receptor is constitutively expressed in the naïve murine cornea. Representative confocal micrographs of naïve BALB/c corneas in peripheral (A–C) and central (D–F) stroma show expression of CD11c + (red, A, D) and CXCR4 + (green, B, E) and colocalization (yellow, C, F) throughout the corneal stroma. Orthogonal views highlighting positive staining added to each corneal area. White dotted boxes show examples of CD11c + DCs (magnified in A–C, stroma (i) and epithelium (ii); and D–F insets), as well as a CD11c − cells (magnified in A–C, inset i) coexpressing CXCR4 in the peripheral and central corneas. Isotype controls for CD11c and CXCR4 do not show positive staining (G–I). Cell density quantification for CD11c + and CXCR4 + cells (cells/mm 2 ) highlights populations of steady-state cDCs expressing CXCR4: significance for cell densities between central and peripheral cornea is denoted by an asterisk (J). Representative histograms of flow cytometry analysis of relative CXCR4 expression of CD45 + leukocytes in the inflamed corneal epithelium (K) and stroma (L). Data are shown as mean ± SEM. t-test, *P

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

29) Product Images from "CD200 Receptor Restriction of Myeloid Cell Responses Antagonizes Antiviral Immunity and Facilitates Cytomegalovirus Persistence within Mucosal Tissue"

Article Title: CD200 Receptor Restriction of Myeloid Cell Responses Antagonizes Antiviral Immunity and Facilitates Cytomegalovirus Persistence within Mucosal Tissue

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004641

CD200 is expressed during in vivo MCMV infection. (A-B) Wt mice were infected with MCMV and SGs harvested 7 days pi. (A) CD31 (green) and CD200 (red) co-localization in SGs. (B) Distinct CD200 (red) expression from EpCAM + (green) acinar epithelial cells (top). Isotype controls for CD200 (Rat IgG2a-Biotin) and EpCAM (Rabbit IgG), and the secondary antibodies Streptavidin 555 and Alexa Fluor 488 anti-rabbit IgG (Bottom). (C) Hematopoietic cell (green [CD45]) expression of CD200 (red). (A-C) Magnification = 63x, white scale bars = 20μm. (D) Representative histogram overlays of CD200 expression by SG-APCs, CD4 and CD8 T cells and NK cells in the SGs at day 7 pi. FMO controls are shown from cells isolated at day 7 pi. (E) CD200 expression by SG and splenic myeloid cell populations was assessed over time. Splenic DCs: CD11c + MHC II + ; splenic macs: F4/80 + CD11b + . Data is represented as mean ± SEM of 3 mice/group representing 4 experiments.
Figure Legend Snippet: CD200 is expressed during in vivo MCMV infection. (A-B) Wt mice were infected with MCMV and SGs harvested 7 days pi. (A) CD31 (green) and CD200 (red) co-localization in SGs. (B) Distinct CD200 (red) expression from EpCAM + (green) acinar epithelial cells (top). Isotype controls for CD200 (Rat IgG2a-Biotin) and EpCAM (Rabbit IgG), and the secondary antibodies Streptavidin 555 and Alexa Fluor 488 anti-rabbit IgG (Bottom). (C) Hematopoietic cell (green [CD45]) expression of CD200 (red). (A-C) Magnification = 63x, white scale bars = 20μm. (D) Representative histogram overlays of CD200 expression by SG-APCs, CD4 and CD8 T cells and NK cells in the SGs at day 7 pi. FMO controls are shown from cells isolated at day 7 pi. (E) CD200 expression by SG and splenic myeloid cell populations was assessed over time. Splenic DCs: CD11c + MHC II + ; splenic macs: F4/80 + CD11b + . Data is represented as mean ± SEM of 3 mice/group representing 4 experiments.

Techniques Used: In Vivo, Infection, Mouse Assay, Expressing, Isolation, Magnetic Cell Separation

30) Product Images from "Endothelial transplantation rejuvenates aged hematopoietic stem cell function"

Article Title: Endothelial transplantation rejuvenates aged hematopoietic stem cell function

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI93940

Aged ECs are sufficient to induce aged hematopoietic phenotypes. ( A and B ) Quantification of hematopoietic expansion by flow cytometry. ( A ) Total phenotypic CD45 + hematopoietic cells and ( B ) CD45 + lineage – cKIT + SCA1 + HSPCs ( n = 3 independent cocultures). ( C ) Quantification of CD45.2 + donor chimerism in PB 4 months after transplantation (Tpx), as measured by flow cytometry ( n = 5 mice/cohort). Results show the ability of young ECs to restore hematopoietic engraftment of HSPCs following coculture, while aged ECs impaired young hematopoietic engraftment relative to that seen in age-matched coculture controls. Unmanipulated pre-expansion WBM cells from young or aged mice were competitively transplanted into lethally irradiated recipients to confirm age-dependent hematopoietic reconstitution phenotypes ( n = 5 mice/cohort). ( D – F ) Quantification of donor-derived lineage + hematopoietic repopulation 4 months after transplantation. Frequencies of ( D ) CD11B + GR1 + myeloid cells, ( E ) B220 + CD19 + B cells, and ( F ) CD8 + (black)/CD4 + (gray) T cell populations in PB were determined by flow cytometry. Young HSPCs cocultured with aged ECs acquired myeloid-biased engraftment at the expense of lymphopoiesis, while young ECs were unable to reverse the myeloid bias in aged HSPC expansions. Error bars represent the sample mean ± SEM. * P
Figure Legend Snippet: Aged ECs are sufficient to induce aged hematopoietic phenotypes. ( A and B ) Quantification of hematopoietic expansion by flow cytometry. ( A ) Total phenotypic CD45 + hematopoietic cells and ( B ) CD45 + lineage – cKIT + SCA1 + HSPCs ( n = 3 independent cocultures). ( C ) Quantification of CD45.2 + donor chimerism in PB 4 months after transplantation (Tpx), as measured by flow cytometry ( n = 5 mice/cohort). Results show the ability of young ECs to restore hematopoietic engraftment of HSPCs following coculture, while aged ECs impaired young hematopoietic engraftment relative to that seen in age-matched coculture controls. Unmanipulated pre-expansion WBM cells from young or aged mice were competitively transplanted into lethally irradiated recipients to confirm age-dependent hematopoietic reconstitution phenotypes ( n = 5 mice/cohort). ( D – F ) Quantification of donor-derived lineage + hematopoietic repopulation 4 months after transplantation. Frequencies of ( D ) CD11B + GR1 + myeloid cells, ( E ) B220 + CD19 + B cells, and ( F ) CD8 + (black)/CD4 + (gray) T cell populations in PB were determined by flow cytometry. Young HSPCs cocultured with aged ECs acquired myeloid-biased engraftment at the expense of lymphopoiesis, while young ECs were unable to reverse the myeloid bias in aged HSPC expansions. Error bars represent the sample mean ± SEM. * P

Techniques Used: Flow Cytometry, Cytometry, Transplantation Assay, Mouse Assay, Irradiation, Derivative Assay

Infusion of young endothelium promotes hematopoietic recovery in aged recipients following myelosuppressive injury. ( A ) Schematic of the EC infusion strategy. ( B and C ) Time course of PB recovery of ( B ) young and ( C ) aged mice following irradiation (6.50 Gy) and infusion of either young ECs, aged ECs, or PBS vehicle control ( n = 5 mice/cohort). The results demonstrated the myeloprotective effect of young EC transplantation following hematopoietic insult in both young and aged recipients, while the result with aged EC transplantation was indistinguishable from that observed in the PBS vehicle-infused controls. ( D – F ) Quantification of CD45.2 + donor chimerism and multilineage engraftment in PB 4 months after donor WBM transplantation as measured by flow cytometry ( n = 5 mice/cohort). ( D ) Unmanipulated steady-state young and aged WBM was competitively transplanted to confirm reduced CD45.2 + hematopoietic engraftment and phenotypic CD11B + /GR1 + myeloid bias in the aged WBM transplantation cohort ( n = 5 mice/cohort). ( E ) Young and ( F ) aged donors infused with young ECs demonstrated an increase in hematopoietic engraftment, while supporting an increase in B220 + and CD3 + lymphoid reconstitution. Error bars represent the sample mean ± SEM. * P
Figure Legend Snippet: Infusion of young endothelium promotes hematopoietic recovery in aged recipients following myelosuppressive injury. ( A ) Schematic of the EC infusion strategy. ( B and C ) Time course of PB recovery of ( B ) young and ( C ) aged mice following irradiation (6.50 Gy) and infusion of either young ECs, aged ECs, or PBS vehicle control ( n = 5 mice/cohort). The results demonstrated the myeloprotective effect of young EC transplantation following hematopoietic insult in both young and aged recipients, while the result with aged EC transplantation was indistinguishable from that observed in the PBS vehicle-infused controls. ( D – F ) Quantification of CD45.2 + donor chimerism and multilineage engraftment in PB 4 months after donor WBM transplantation as measured by flow cytometry ( n = 5 mice/cohort). ( D ) Unmanipulated steady-state young and aged WBM was competitively transplanted to confirm reduced CD45.2 + hematopoietic engraftment and phenotypic CD11B + /GR1 + myeloid bias in the aged WBM transplantation cohort ( n = 5 mice/cohort). ( E ) Young and ( F ) aged donors infused with young ECs demonstrated an increase in hematopoietic engraftment, while supporting an increase in B220 + and CD3 + lymphoid reconstitution. Error bars represent the sample mean ± SEM. * P

Techniques Used: Mouse Assay, Irradiation, Transplantation Assay, Flow Cytometry, Cytometry

Young EC coinfusion radioprotects the BM microenvironment. Lethally irradiated (9.50 Gy) mice were coinfused with either 10 5 young or aged WBM cells and 5 × 10 5 young BM ECs. ( A ) Representative H E-stained longitudinal femur sections from coinfused mice 7 days after irradiation ( n = 10 mice/cohort). Original magnification, ×100. ( B ) Representative images of damaged VEGFR3 + femoral vessels, including type I hemorrhagic (asterisk), type I discontinuous (red arrow), and type II regressed (blue arrow), 7 days after irradiation, demonstrating radioprotection of the vascular niche ( n = 10 mice/cohort). Sections were counterstained with hematoxylin. Original magnification, ×200. ( C and D ) Quantification of total BM CD45 + cells showing mitigation of panhematopoietic injury in cohorts coinfused with young or aged WBM and young ECs ( n = 10 mice/cohort; data are related to A ). ( E and F ) Quantification of type I/II damaged VEGFR3 + sinusoidal vessels in cohorts coinfused with young or aged WBM and young ECs ( n = 10 mice/cohort; data are related to B ). Error bars represent the sample mean ± SEM. *** P
Figure Legend Snippet: Young EC coinfusion radioprotects the BM microenvironment. Lethally irradiated (9.50 Gy) mice were coinfused with either 10 5 young or aged WBM cells and 5 × 10 5 young BM ECs. ( A ) Representative H E-stained longitudinal femur sections from coinfused mice 7 days after irradiation ( n = 10 mice/cohort). Original magnification, ×100. ( B ) Representative images of damaged VEGFR3 + femoral vessels, including type I hemorrhagic (asterisk), type I discontinuous (red arrow), and type II regressed (blue arrow), 7 days after irradiation, demonstrating radioprotection of the vascular niche ( n = 10 mice/cohort). Sections were counterstained with hematoxylin. Original magnification, ×200. ( C and D ) Quantification of total BM CD45 + cells showing mitigation of panhematopoietic injury in cohorts coinfused with young or aged WBM and young ECs ( n = 10 mice/cohort; data are related to A ). ( E and F ) Quantification of type I/II damaged VEGFR3 + sinusoidal vessels in cohorts coinfused with young or aged WBM and young ECs ( n = 10 mice/cohort; data are related to B ). Error bars represent the sample mean ± SEM. *** P

Techniques Used: Irradiation, Mouse Assay, Staining

Aged BM vasculature displays functional alterations in vivo. ( A ) Representative longitudinal and inset images of femurs intravitally labeled with a vascular-specific VECAD antibody (red), showing morphological alterations in aged vasculature (white line demarcates cortical bone). Scale bars: 100 μm (longitudinal images) and 50 μm (insets). ( B and C ) Analysis of BM vascular leakiness in young and aged femurs. ( B ) Quantification of Evans blue dye extravasation ( n = 5 mice/cohort). ( C ) Representative femurs injected with Evans blue dye. Noninjected controls were used to determine baselines ( n = 5 mice/cohort). ( D and E ) Frequency of recoverable ( D ) VECAD + CD31 + CD45 – TER119 – BM ECs and ( E ) VECAD – CD31 – CD45 – TER119 – stroma in young and aged femurs ( n = 5 mice/cohort). ( F ) Quantification of mean fluorescence intensity (MFI) and representative histogram of ROS in VECAD + CD31 + CD45 – TER119 – ECs from young and aged femurs showing an increase in ROS in aged ECs ( n = 3 mice/cohort). ( G ) MFI quantification and representative histogram of pimonidazole adducts as detected by an anti-pimonidazole antibody (HypoxyProbe) in VECAD + CD31 + CD45 – TER119 – ECs from young and aged femurs, demonstrating an increased hypoxia state in aged ECs ( n = 3 mice/cohort). ( H ) Representative immunofluorescence images of HypoxyProbe-stained young and aged femurs, showing local changes in hypoxia (white line demarcates cortical bone). Scale bar: 50 μm. Error bars represent the sample mean ± SEM. * P
Figure Legend Snippet: Aged BM vasculature displays functional alterations in vivo. ( A ) Representative longitudinal and inset images of femurs intravitally labeled with a vascular-specific VECAD antibody (red), showing morphological alterations in aged vasculature (white line demarcates cortical bone). Scale bars: 100 μm (longitudinal images) and 50 μm (insets). ( B and C ) Analysis of BM vascular leakiness in young and aged femurs. ( B ) Quantification of Evans blue dye extravasation ( n = 5 mice/cohort). ( C ) Representative femurs injected with Evans blue dye. Noninjected controls were used to determine baselines ( n = 5 mice/cohort). ( D and E ) Frequency of recoverable ( D ) VECAD + CD31 + CD45 – TER119 – BM ECs and ( E ) VECAD – CD31 – CD45 – TER119 – stroma in young and aged femurs ( n = 5 mice/cohort). ( F ) Quantification of mean fluorescence intensity (MFI) and representative histogram of ROS in VECAD + CD31 + CD45 – TER119 – ECs from young and aged femurs showing an increase in ROS in aged ECs ( n = 3 mice/cohort). ( G ) MFI quantification and representative histogram of pimonidazole adducts as detected by an anti-pimonidazole antibody (HypoxyProbe) in VECAD + CD31 + CD45 – TER119 – ECs from young and aged femurs, demonstrating an increased hypoxia state in aged ECs ( n = 3 mice/cohort). ( H ) Representative immunofluorescence images of HypoxyProbe-stained young and aged femurs, showing local changes in hypoxia (white line demarcates cortical bone). Scale bar: 50 μm. Error bars represent the sample mean ± SEM. * P

Techniques Used: Functional Assay, In Vivo, Labeling, Mouse Assay, Injection, Fluorescence, Immunofluorescence, Staining

31) Product Images from "TGFβ1 neutralization displays therapeutic efficacy through both an immunomodulatory and a non-immune tumor-intrinsic mechanism"

Article Title: TGFβ1 neutralization displays therapeutic efficacy through both an immunomodulatory and a non-immune tumor-intrinsic mechanism

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2020-001798

Expression of TGFβ isoforms and EMT-associated genes in TiRP melanoma cells and stromal cells. (A) Separation of tumor cells and stromal cells from Mela and Amela TiRP tumors. CD45-negative cells were sorted based on P1A expression using a fluorescently labeled P1A-mRNA SmartFlare probe. Sorted cells were verified by staining with P1A-specific antibody P1A102B3. (B) Cells sorted in (A) were tested by RT-qPCR for the indicated genes. Results are expressed as mean number of transcripts±SEM, from a total 35 Mela and 110 Amela tumors tested. EMT, epithelial-to-mesenchymal transition; TGFβ, transforming growth factor-β.
Figure Legend Snippet: Expression of TGFβ isoforms and EMT-associated genes in TiRP melanoma cells and stromal cells. (A) Separation of tumor cells and stromal cells from Mela and Amela TiRP tumors. CD45-negative cells were sorted based on P1A expression using a fluorescently labeled P1A-mRNA SmartFlare probe. Sorted cells were verified by staining with P1A-specific antibody P1A102B3. (B) Cells sorted in (A) were tested by RT-qPCR for the indicated genes. Results are expressed as mean number of transcripts±SEM, from a total 35 Mela and 110 Amela tumors tested. EMT, epithelial-to-mesenchymal transition; TGFβ, transforming growth factor-β.

Techniques Used: Expressing, Labeling, Staining, Quantitative RT-PCR

32) Product Images from "Chemotherapy accelerates immune-senescence and functional impairments of Vδ2pos T cells in elderly patients affected by liver metastatic colorectal cancer"

Article Title: Chemotherapy accelerates immune-senescence and functional impairments of Vδ2pos T cells in elderly patients affected by liver metastatic colorectal cancer

Journal: Journal for Immunotherapy of Cancer

doi: 10.1186/s40425-019-0825-4

Frequency and distributions of peripheral blood Vδ2 pos T cell subsets in patients affected by liver metastasis of colorectal cancer and underwent chemotherapy. a Representative dot plot flow cytometric graphs showing the gating strategy of viable CD45 pos /CD3 pos /Vδ2 pos T lymphocytes. b Statistical dot plot graph showing the absolute number of CD3 pos (left) and Vδ2 pos (right) T cells per 1 mL of blood in healthy donors ( n = 12; mean age: 49.3 ± 9.5 ) and CLM patients underwent CHT regimens ( n = 16; mean age: 51.5 ± 8.1 ). c - e Representative dot plot graph flow cytometric graph ( c ) and pie charts ( d and e ) showing respectively the distribution and the percentages of CD27 pos /CD45RA pos T Naive (upper right in dot plot graph and light green in pie charts), CD27 pos /CD45RA neg central memory (T CM ) (upper left in dot plot graph and gray in pie charts), CD27 neg /CD45RA neg effector-memory (T EM ) (lower left in dot plot graph and purple in pie charts) and terminally-differentiated CD27 neg /CD45RA pos (T EMRA ) (lower right in dot plot graph and orange in pie charts) Vδ2 pos T cell subsets. Pie charts compare the frequencies of Vδ2 pos T cell subsets between healthy donors ( n = 34; mean age: 51.7 ± 10.8 ) with age-matched CLM patient underwent CHT ( n = 33; mean age: 51.5 ± 8.1 ) d as well as between CLM patients naïve for CHT ( n = 13; mean age: 69.5 ± 8.1 ) and age-matched CLM patients underwent CHT ( n = 41; mean age: 70.1 ± 6.5 ) ( e ). f Statistical analysis showing the Pearson correlations between the frequency (%) of either T CM (left) or T EMRA (right) Vδ2 pos T cells with the number of CHT cycles ( mean number: 8.7 ± 6.5 ) administered to patients affected by CLM ( n = 40 )
Figure Legend Snippet: Frequency and distributions of peripheral blood Vδ2 pos T cell subsets in patients affected by liver metastasis of colorectal cancer and underwent chemotherapy. a Representative dot plot flow cytometric graphs showing the gating strategy of viable CD45 pos /CD3 pos /Vδ2 pos T lymphocytes. b Statistical dot plot graph showing the absolute number of CD3 pos (left) and Vδ2 pos (right) T cells per 1 mL of blood in healthy donors ( n = 12; mean age: 49.3 ± 9.5 ) and CLM patients underwent CHT regimens ( n = 16; mean age: 51.5 ± 8.1 ). c - e Representative dot plot graph flow cytometric graph ( c ) and pie charts ( d and e ) showing respectively the distribution and the percentages of CD27 pos /CD45RA pos T Naive (upper right in dot plot graph and light green in pie charts), CD27 pos /CD45RA neg central memory (T CM ) (upper left in dot plot graph and gray in pie charts), CD27 neg /CD45RA neg effector-memory (T EM ) (lower left in dot plot graph and purple in pie charts) and terminally-differentiated CD27 neg /CD45RA pos (T EMRA ) (lower right in dot plot graph and orange in pie charts) Vδ2 pos T cell subsets. Pie charts compare the frequencies of Vδ2 pos T cell subsets between healthy donors ( n = 34; mean age: 51.7 ± 10.8 ) with age-matched CLM patient underwent CHT ( n = 33; mean age: 51.5 ± 8.1 ) d as well as between CLM patients naïve for CHT ( n = 13; mean age: 69.5 ± 8.1 ) and age-matched CLM patients underwent CHT ( n = 41; mean age: 70.1 ± 6.5 ) ( e ). f Statistical analysis showing the Pearson correlations between the frequency (%) of either T CM (left) or T EMRA (right) Vδ2 pos T cells with the number of CHT cycles ( mean number: 8.7 ± 6.5 ) administered to patients affected by CLM ( n = 40 )

Techniques Used: Flow Cytometry

33) Product Images from "IgA transcytosis and antigen recognition govern ovarian cancer immunity"

Article Title: IgA transcytosis and antigen recognition govern ovarian cancer immunity

Journal: Nature

doi: 10.1038/s41586-020-03144-0

Tumour-derived IgA abrogates tumour growth through antigen-dependent redirection of Fcα/μR + myeloid cells and antigen-independent, pIgR-mediated transcytosis. a , Dot plots showing FACS analysis of splenocytes for NK1.1 depletion in RAG1-KO mice (left). Scatter plot showing CD45 + NK1.1 + cells percentages among viable splenocytes in respective treatment-group mice ( n = 10 mice per group pooled from 2 independent experiments) (right). P
Figure Legend Snippet: Tumour-derived IgA abrogates tumour growth through antigen-dependent redirection of Fcα/μR + myeloid cells and antigen-independent, pIgR-mediated transcytosis. a , Dot plots showing FACS analysis of splenocytes for NK1.1 depletion in RAG1-KO mice (left). Scatter plot showing CD45 + NK1.1 + cells percentages among viable splenocytes in respective treatment-group mice ( n = 10 mice per group pooled from 2 independent experiments) (right). P

Techniques Used: Derivative Assay, FACS, Mouse Assay

IgA–pIgR colocalization is associated with protective immunity in human ovarian cancer. a , Left, percentage of FACS cell counts of IgA + , IgG + or IgM + cells among Ig + B cells or plasmablasts or plasma cells, normalized to 10,000 viable CD45 + cells. B cells, CD45 + CD3 − CD19 + CD20 + cells; plasmablasts, CD45 + CD3 − CD19 + CD20 − CD38 high cells; plasma cells, CD45 + CD3 − CD19 + CD20 − CD138 + and CD45 + CD3 − CD19 − CD20 − CD138 + cells. Each dot represents one tumour ( n = 29). Details of box plots can be found in Methods. P values were obtained by a two-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. Supplementary Table 1 provides further details on statistics. Right, bar graphs representing the percentage of each isotype produced by plasma cells (top) or B cells (bottom) in the same tumours, normalized to 10,000 viable CD45 + cells. IC, intracellular. b , IgA-coated CD45 − EpCAM + tumour epithelial cells (mean ± s.e.m., n = 10) in dissociated HGSOC. c , Expression of pIgR protein in independent HGSOC ( n = 27); tumour-free Fallopian tube ( n = 3), ovary ( n = 5) and omental ( n = 4) samples; ovarian tumour cell lines; and K562 leukaemia cells and THP1 monocyte cells (negative controls). Positive control, recombinant human pIgR. Western blots were repeated twice. NSCLC, non-small-cell lung cancer. d , Histograms showing FACS analysis of pIgR, in ovarian surface epithelial (OSE), K562, THP1, wild-type or PIGR -ablated (pIgR CRISPR ) OVCAR3 cells. e , Left, representative ( n = 273) combined staining of IgA, pIgR, IgG, PCK and DAPI. Instances with IgA–pIgR colocalization are indicated with arrows. Scale bar, 50 μm (top left), 20 μm (all other panels). Top right, representative ( n = 137, IgA–pIgR colocalization ≥ median) dot plot showing IgA–pIgR colocalized signal among DAPI + PCK + cells. Bottom right, scattered graph showing number of IgA–pIgR colocalized cells (averaged from duplicated cores) per mm 2 of PCK + (mean ± s.e.m., n = 273). f , Increased numbers of cells with IgA–pIgR colocalization per PCK + tumour islet area (averaged from duplicated cores) are associated with improved outcome (threshold, median; P = 0.0116, H. Lee Moffett Cancer Centre cohort (MCC) (right); P = 0.0002, New England Case–Control study cohort (NECC) (left)). g , Density of IgA-coated cells (averaged from duplicated cores) in tumour islets (cells per mm 2 PCK + area) is associated with improved outcome ( P = 0.0110 for MCC (right) and P = 0.0054 for NECC (left) cohorts). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, two-sided log-rank (Mantel–Cox) test. Source data
Figure Legend Snippet: IgA–pIgR colocalization is associated with protective immunity in human ovarian cancer. a , Left, percentage of FACS cell counts of IgA + , IgG + or IgM + cells among Ig + B cells or plasmablasts or plasma cells, normalized to 10,000 viable CD45 + cells. B cells, CD45 + CD3 − CD19 + CD20 + cells; plasmablasts, CD45 + CD3 − CD19 + CD20 − CD38 high cells; plasma cells, CD45 + CD3 − CD19 + CD20 − CD138 + and CD45 + CD3 − CD19 − CD20 − CD138 + cells. Each dot represents one tumour ( n = 29). Details of box plots can be found in Methods. P values were obtained by a two-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. Supplementary Table 1 provides further details on statistics. Right, bar graphs representing the percentage of each isotype produced by plasma cells (top) or B cells (bottom) in the same tumours, normalized to 10,000 viable CD45 + cells. IC, intracellular. b , IgA-coated CD45 − EpCAM + tumour epithelial cells (mean ± s.e.m., n = 10) in dissociated HGSOC. c , Expression of pIgR protein in independent HGSOC ( n = 27); tumour-free Fallopian tube ( n = 3), ovary ( n = 5) and omental ( n = 4) samples; ovarian tumour cell lines; and K562 leukaemia cells and THP1 monocyte cells (negative controls). Positive control, recombinant human pIgR. Western blots were repeated twice. NSCLC, non-small-cell lung cancer. d , Histograms showing FACS analysis of pIgR, in ovarian surface epithelial (OSE), K562, THP1, wild-type or PIGR -ablated (pIgR CRISPR ) OVCAR3 cells. e , Left, representative ( n = 273) combined staining of IgA, pIgR, IgG, PCK and DAPI. Instances with IgA–pIgR colocalization are indicated with arrows. Scale bar, 50 μm (top left), 20 μm (all other panels). Top right, representative ( n = 137, IgA–pIgR colocalization ≥ median) dot plot showing IgA–pIgR colocalized signal among DAPI + PCK + cells. Bottom right, scattered graph showing number of IgA–pIgR colocalized cells (averaged from duplicated cores) per mm 2 of PCK + (mean ± s.e.m., n = 273). f , Increased numbers of cells with IgA–pIgR colocalization per PCK + tumour islet area (averaged from duplicated cores) are associated with improved outcome (threshold, median; P = 0.0116, H. Lee Moffett Cancer Centre cohort (MCC) (right); P = 0.0002, New England Case–Control study cohort (NECC) (left)). g , Density of IgA-coated cells (averaged from duplicated cores) in tumour islets (cells per mm 2 PCK + area) is associated with improved outcome ( P = 0.0110 for MCC (right) and P = 0.0054 for NECC (left) cohorts). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, two-sided log-rank (Mantel–Cox) test. Source data

Techniques Used: FACS, Produced, Expressing, Positive Control, Recombinant, Western Blot, CRISPR, Staining

IgA coating of tumour cells is associated with a better outcome in HGSOC. a , Bar graphs representing tumour-wise FACS analysis ( n = 10) comparison of percentages of each Ig + cells among total Ig + plasmablasts (intracellular in CD45 + CD3 − CD19 + CD20 − CD38 high ) and CD27 + plasmablasts (intracellular in CD45 + CD3 − CD19 + CD20 − CD38 high CD27 + ), normalized to 10,000 viable CD45 + cells. b , Relative abundances of IgH chains on the basis of TCGA transcriptional analyses ( n = 430) for each immunoglobulin heavy chain gene. Abundances are shown in log 2 -transformed reads per kilobase of transcripts (RPKM) values, which corrects for both gene length and sequencing depth. Details of box plots are in Methods. c , Overall survival associated with the presence of CD19 + CD138 + plasma cells within the total tumour area ( P = 0.0285) (left) or specifically in the PCK + epithelial tumour islets ( P = 0.0053) (right), in HGSOC as assessed by multiplex immunohistochemistry of TMAs corresponding to 534 patients with HGSOC combined from the NECC ( n = 180), NHS ( n = 261) and MCC ( n = 93). Plasma cell infiltration is defined as the presence of CD19 + CD138 + cells on any of the duplicate sections analysed for each tumour. * P ≤ 0.05, ** P ≤ 0.01, two-sided log-rank (Mantel–Cox) test. d , FACS analysis showing number (log-transformed) of plasma cells (CD45 + CD3 − CD19 + CD20 − CD138 + and CD45 + CD3 − CD19 − CD20 − CD138 + ), plasmablasts (CD45 + CD3 − CD19 + CD20 − CD38 high ), B cells (CD45 + CD3 − CD19 + CD20 + ), T cells (CD45 + CD3 + ) and other leukocytes (CD45 + CD3 − CD19 − CD20 − CD138 − ) in HGSOC ( n = 29). The data are normalized to 10,000 viable CD45 + leukocytes. Data are mean ± s.e.m. e , Graphs showing correlations between log count of T cells and plasma cells (left) (Pearson correlation coefficient ( r ) = 0.5049; two-sided nominal P = 0.0052); and between log count of T cells and plasmablasts (right) (Pearson correlation coefficient ( r ) = 0.4755; two-sided nominal P = 0.0091). All three cell types represent absolute counts normalized to 10,000 CD45 + leukocytes. f , Colocalization of IgA with pIgR + cells (IgA–pIgR co-localization ≥ median) in the PCK + tumour islets is associated with an improved outcome in HGSOC, compared to only pIgR high samples (≥median) without IgA colocalization (colocalization
Figure Legend Snippet: IgA coating of tumour cells is associated with a better outcome in HGSOC. a , Bar graphs representing tumour-wise FACS analysis ( n = 10) comparison of percentages of each Ig + cells among total Ig + plasmablasts (intracellular in CD45 + CD3 − CD19 + CD20 − CD38 high ) and CD27 + plasmablasts (intracellular in CD45 + CD3 − CD19 + CD20 − CD38 high CD27 + ), normalized to 10,000 viable CD45 + cells. b , Relative abundances of IgH chains on the basis of TCGA transcriptional analyses ( n = 430) for each immunoglobulin heavy chain gene. Abundances are shown in log 2 -transformed reads per kilobase of transcripts (RPKM) values, which corrects for both gene length and sequencing depth. Details of box plots are in Methods. c , Overall survival associated with the presence of CD19 + CD138 + plasma cells within the total tumour area ( P = 0.0285) (left) or specifically in the PCK + epithelial tumour islets ( P = 0.0053) (right), in HGSOC as assessed by multiplex immunohistochemistry of TMAs corresponding to 534 patients with HGSOC combined from the NECC ( n = 180), NHS ( n = 261) and MCC ( n = 93). Plasma cell infiltration is defined as the presence of CD19 + CD138 + cells on any of the duplicate sections analysed for each tumour. * P ≤ 0.05, ** P ≤ 0.01, two-sided log-rank (Mantel–Cox) test. d , FACS analysis showing number (log-transformed) of plasma cells (CD45 + CD3 − CD19 + CD20 − CD138 + and CD45 + CD3 − CD19 − CD20 − CD138 + ), plasmablasts (CD45 + CD3 − CD19 + CD20 − CD38 high ), B cells (CD45 + CD3 − CD19 + CD20 + ), T cells (CD45 + CD3 + ) and other leukocytes (CD45 + CD3 − CD19 − CD20 − CD138 − ) in HGSOC ( n = 29). The data are normalized to 10,000 viable CD45 + leukocytes. Data are mean ± s.e.m. e , Graphs showing correlations between log count of T cells and plasma cells (left) (Pearson correlation coefficient ( r ) = 0.5049; two-sided nominal P = 0.0052); and between log count of T cells and plasmablasts (right) (Pearson correlation coefficient ( r ) = 0.4755; two-sided nominal P = 0.0091). All three cell types represent absolute counts normalized to 10,000 CD45 + leukocytes. f , Colocalization of IgA with pIgR + cells (IgA–pIgR co-localization ≥ median) in the PCK + tumour islets is associated with an improved outcome in HGSOC, compared to only pIgR high samples (≥median) without IgA colocalization (colocalization

Techniques Used: FACS, Transformation Assay, Sequencing, Multiplex Assay, Immunohistochemistry

Transcytosis of IgA through pIgR + ovarian cancer cells impairs tumour growth and augments cytotoxic killing mediated by T cells. a , Left, images of APC-labelled IgA binding and internalization in pIgR + OVCAR3 cells (repeated three times). Scale bar, 50 μm (main panels), 10 μm (magnified regions). Right, comparison of antibody internalization signal (mean ± s.e.m.) in different treatment conditions and at different temporal points. Each dot represents quantification from one cell. *** P ≤ 0.001, unpaired two-tailed t -test. Supplementary Table 2 provides details of statistics. b , OVCAR3 cells were incubated with control IgA or IgG for 8 h in the presence of wortmannin, brefeldin A (BFA) or vehicle, and supernatants were subjected to liquid chromatography with tandem mass spectrometry (LC–MS/MS). Heat map of all peptides of the extracellular domain of pIgR ( n = 3); scale represents log 2 -transformed intensities of pIgR peptide fragments detected in LC–MS/MS. c , Left, co-immunoprecipitates of supernatants from IgA-treated pIgR + or PIGR -ablated OVCAR3 cells (with and without brefeldin A or wortmannin) blotted for the secretory component of pIgR and IgA (input control). Right, LC–MS/MS analysis of the co-immunoprecipitates showing intensities (log 2 -transformed) of the secretory component of pIgR and IgA ( n = 2). WT, wild type; CR, PIGR -ablated. d , Pre-ranked gene-set enrichment analysis (GSEA), showing the top upregulated gene sets in OVCAR3 cells treated with irrelevant IgA compared to IgG or untreated cells ( n = 3), Kolmogorov–Smirnov test. GO, Gene Ontology. e , Progressive increase in DUSP5 and concomitant reduction in phospho-ERK1 and phospho-ERK2 (pERK1/2) after IgA treatment (left) of OVCAR3 cells, but not IgG treatment (right). Experiments were repeated three times. tERK1/2, total ERK1 and total ERK2. f , Left, dose-dependent cytotoxic killing of NY-ESO-1-transduced OVCAR3 cells ( n = 3) by NY-ESO-1–TCR-transduced T cells is augmented by co-incubation with IgA, compared to IgG or PBS. Right, IgA treatment also augmented the anti-tumour activity of FSH-targeted chimeric receptor T cells. Mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ordinary one-way ANOVA. Supplementary Table 2 provides details of statistics. g , Cytotoxic killing of primary CD45 − EpCAM + tumour cells ( n = 3) by autologous tumour-infiltrating T cells (1:1 ratio) is augmented by co-incubation with autologous ( P
Figure Legend Snippet: Transcytosis of IgA through pIgR + ovarian cancer cells impairs tumour growth and augments cytotoxic killing mediated by T cells. a , Left, images of APC-labelled IgA binding and internalization in pIgR + OVCAR3 cells (repeated three times). Scale bar, 50 μm (main panels), 10 μm (magnified regions). Right, comparison of antibody internalization signal (mean ± s.e.m.) in different treatment conditions and at different temporal points. Each dot represents quantification from one cell. *** P ≤ 0.001, unpaired two-tailed t -test. Supplementary Table 2 provides details of statistics. b , OVCAR3 cells were incubated with control IgA or IgG for 8 h in the presence of wortmannin, brefeldin A (BFA) or vehicle, and supernatants were subjected to liquid chromatography with tandem mass spectrometry (LC–MS/MS). Heat map of all peptides of the extracellular domain of pIgR ( n = 3); scale represents log 2 -transformed intensities of pIgR peptide fragments detected in LC–MS/MS. c , Left, co-immunoprecipitates of supernatants from IgA-treated pIgR + or PIGR -ablated OVCAR3 cells (with and without brefeldin A or wortmannin) blotted for the secretory component of pIgR and IgA (input control). Right, LC–MS/MS analysis of the co-immunoprecipitates showing intensities (log 2 -transformed) of the secretory component of pIgR and IgA ( n = 2). WT, wild type; CR, PIGR -ablated. d , Pre-ranked gene-set enrichment analysis (GSEA), showing the top upregulated gene sets in OVCAR3 cells treated with irrelevant IgA compared to IgG or untreated cells ( n = 3), Kolmogorov–Smirnov test. GO, Gene Ontology. e , Progressive increase in DUSP5 and concomitant reduction in phospho-ERK1 and phospho-ERK2 (pERK1/2) after IgA treatment (left) of OVCAR3 cells, but not IgG treatment (right). Experiments were repeated three times. tERK1/2, total ERK1 and total ERK2. f , Left, dose-dependent cytotoxic killing of NY-ESO-1-transduced OVCAR3 cells ( n = 3) by NY-ESO-1–TCR-transduced T cells is augmented by co-incubation with IgA, compared to IgG or PBS. Right, IgA treatment also augmented the anti-tumour activity of FSH-targeted chimeric receptor T cells. Mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ordinary one-way ANOVA. Supplementary Table 2 provides details of statistics. g , Cytotoxic killing of primary CD45 − EpCAM + tumour cells ( n = 3) by autologous tumour-infiltrating T cells (1:1 ratio) is augmented by co-incubation with autologous ( P

Techniques Used: Binding Assay, Two Tailed Test, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Activity Assay

IgA transcytosis through HGSOC cells has substantial anti-tumour effects and sensitizes tumour cells to cytolytic killing by T cells. a , Immunoblots showing pIgR co-immunoprecipitation with IgA, using nondenaturing lysates from two HGSOCs, CD45 + and CD45 − cells isolated from human ovarian cancer ascites, ascitic fluid and OVCAR3 cells (negative control). Inputs were immunoblotted using 1% of the amount of lysate used for the co-immunoprecipitation. The experiments were repeated three times. b , OVCAR3 cells were incubated with 0.5 μg ml −1 of control IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were subjected to mass spectrometry. The amino acids 62–77 fragment of pIgR was found only after incubation with IgA (repeated three times). c–e , OVCAR4 ( c ), OVCAR5 ( d ) and primary HGSOC ( e ) tumour cells were incubated with 0.5 μg ml −1 of irrelevant IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were then subjected to mass spectrometry (left). Right, heat map of all peptides of the extracellular domain of pIgR ( n = 3). BFA, brefeldin A. WM, wortmannin. f , g , GSEA enrichment plots ( h ) and heat maps ( g ) using normalized gene expression from RNA-seq analysis from OVCAR3 cells with irrelevant IgA (0.5 μg ml −1 ), IgG (0.5 μg ml −1 ) or no treatment for 24 h ( n = 3 experiments). h , No change in the protein levels of DUSP5, total ERK1/2 or phospho-ERK1/2 after vehicle (PBS) treatment of pIgR + (WT) OVACR3 cells (left) or after IgA treatment of PIGR -ablated OVACR3 cells (right), incubated up to 8 h. The experiments were repeated three times. i , Dose-dependent cytotoxic killing of OVCAR3 cells by FSH-targeted chimeric receptor T cells is augmented by co-incubation with 0.5 μg ml −1 of irrelevant IgA, anti-TSPAN7–IgA or anti-BDNF–IgA compared to IgG, pepsinized irrelevant IgA or PBS. n = 3 per group. ** P ≤ 0.01, unpaired two-tailed t -test. j , Cytotoxic killing of autologous CD45 − EpCAM + tumour cells (with corresponding decrease of annexin V − propidium iodide (PI) − viable cells) by autologous T cells (added at 1:1 ratio) is augmented by co-incubation with 0.5 μg ml −1 of autologous IgA or irrelevant IgA but not with autologous IgG, pepsinized autologous or irrelevant IgA, as compared to uncoated cells ( n = 3). Annexin V + : irrelevant IgA versus pepsinized irrelevant IgA, P
Figure Legend Snippet: IgA transcytosis through HGSOC cells has substantial anti-tumour effects and sensitizes tumour cells to cytolytic killing by T cells. a , Immunoblots showing pIgR co-immunoprecipitation with IgA, using nondenaturing lysates from two HGSOCs, CD45 + and CD45 − cells isolated from human ovarian cancer ascites, ascitic fluid and OVCAR3 cells (negative control). Inputs were immunoblotted using 1% of the amount of lysate used for the co-immunoprecipitation. The experiments were repeated three times. b , OVCAR3 cells were incubated with 0.5 μg ml −1 of control IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were subjected to mass spectrometry. The amino acids 62–77 fragment of pIgR was found only after incubation with IgA (repeated three times). c–e , OVCAR4 ( c ), OVCAR5 ( d ) and primary HGSOC ( e ) tumour cells were incubated with 0.5 μg ml −1 of irrelevant IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were then subjected to mass spectrometry (left). Right, heat map of all peptides of the extracellular domain of pIgR ( n = 3). BFA, brefeldin A. WM, wortmannin. f , g , GSEA enrichment plots ( h ) and heat maps ( g ) using normalized gene expression from RNA-seq analysis from OVCAR3 cells with irrelevant IgA (0.5 μg ml −1 ), IgG (0.5 μg ml −1 ) or no treatment for 24 h ( n = 3 experiments). h , No change in the protein levels of DUSP5, total ERK1/2 or phospho-ERK1/2 after vehicle (PBS) treatment of pIgR + (WT) OVACR3 cells (left) or after IgA treatment of PIGR -ablated OVACR3 cells (right), incubated up to 8 h. The experiments were repeated three times. i , Dose-dependent cytotoxic killing of OVCAR3 cells by FSH-targeted chimeric receptor T cells is augmented by co-incubation with 0.5 μg ml −1 of irrelevant IgA, anti-TSPAN7–IgA or anti-BDNF–IgA compared to IgG, pepsinized irrelevant IgA or PBS. n = 3 per group. ** P ≤ 0.01, unpaired two-tailed t -test. j , Cytotoxic killing of autologous CD45 − EpCAM + tumour cells (with corresponding decrease of annexin V − propidium iodide (PI) − viable cells) by autologous T cells (added at 1:1 ratio) is augmented by co-incubation with 0.5 μg ml −1 of autologous IgA or irrelevant IgA but not with autologous IgG, pepsinized autologous or irrelevant IgA, as compared to uncoated cells ( n = 3). Annexin V + : irrelevant IgA versus pepsinized irrelevant IgA, P

Techniques Used: Western Blot, Immunoprecipitation, Isolation, Negative Control, Incubation, Mass Spectrometry, Expressing, RNA Sequencing Assay, Two Tailed Test

34) Product Images from "Effective recovery of highly purified CD326+ tumor cells from lavage fluid of patients treated with a novel cell-free and concentrated ascites reinfusion therapy (KM-CART)"

Article Title: Effective recovery of highly purified CD326+ tumor cells from lavage fluid of patients treated with a novel cell-free and concentrated ascites reinfusion therapy (KM-CART)

Journal: SpringerPlus

doi: 10.1186/s40064-015-1508-3

Collection of cell components from lavage fluid. Total cellular components were separated from KM-CART lavage fluid by centrifugation, enzymatic digestion, RBC-burst, polymyxin B treatment and filtration (separation steps) followed by CD45 − depletion (enrichment step). a Wright–Giemsa-stained cytocentrifuge preparations are shown. Bars 40 μm. b The mean obtained total cell numbers. After the separation steps: 7.50 × 10 7 cells, and after the enrichment step: 6.39 × 10 6 cells
Figure Legend Snippet: Collection of cell components from lavage fluid. Total cellular components were separated from KM-CART lavage fluid by centrifugation, enzymatic digestion, RBC-burst, polymyxin B treatment and filtration (separation steps) followed by CD45 − depletion (enrichment step). a Wright–Giemsa-stained cytocentrifuge preparations are shown. Bars 40 μm. b The mean obtained total cell numbers. After the separation steps: 7.50 × 10 7 cells, and after the enrichment step: 6.39 × 10 6 cells

Techniques Used: Centrifugation, Filtration, Staining

Protein contents extraction from tumor cells. Protein contents were extracted from enriched tumor cells by the freeze and thaw method. a Recovery efficiency of WBC (CD45 + ) and tumor cells (CD45 − /EpCAM + ). b Protein yield from total ascites
Figure Legend Snippet: Protein contents extraction from tumor cells. Protein contents were extracted from enriched tumor cells by the freeze and thaw method. a Recovery efficiency of WBC (CD45 + ) and tumor cells (CD45 − /EpCAM + ). b Protein yield from total ascites

Techniques Used:

Enrichment of tumor cells for tumor lysate. a A typical dotplot pattern of post-separation and post-enrichment cells. Cells were stained with monoclonal antibodies and were defined as CD45 + CD326 − ; the tumor cells were CD45 − CD326 + . b The frequencies of CD45 + cells (WBC). c The frequencies of CD45 − /EpCAM + cells (tumor cells). d The cell number of CD45 + cells (WBC). e The cell number of CD45 − /EpCAM + cells (tumor cells)
Figure Legend Snippet: Enrichment of tumor cells for tumor lysate. a A typical dotplot pattern of post-separation and post-enrichment cells. Cells were stained with monoclonal antibodies and were defined as CD45 + CD326 − ; the tumor cells were CD45 − CD326 + . b The frequencies of CD45 + cells (WBC). c The frequencies of CD45 − /EpCAM + cells (tumor cells). d The cell number of CD45 + cells (WBC). e The cell number of CD45 − /EpCAM + cells (tumor cells)

Techniques Used: Staining

Schematic diagrams of the KM-CART tumor cell-recovery system. Cellular components (blood cells, tumor cells, etc.) are collected by the KM-CART system, and then the separation and enrichment of tumor cells (CD45 − /EpCAM + ) is performed. a Processing of ascites. b Processing of lavage fluid
Figure Legend Snippet: Schematic diagrams of the KM-CART tumor cell-recovery system. Cellular components (blood cells, tumor cells, etc.) are collected by the KM-CART system, and then the separation and enrichment of tumor cells (CD45 − /EpCAM + ) is performed. a Processing of ascites. b Processing of lavage fluid

Techniques Used:

35) Product Images from "Mesenchymal stem cells loaded on 3D-printed gradient poly(ε-caprolactone)/methacrylated alginate composite scaffolds for cartilage tissue engineering"

Article Title: Mesenchymal stem cells loaded on 3D-printed gradient poly(ε-caprolactone)/methacrylated alginate composite scaffolds for cartilage tissue engineering

Journal: Regenerative Biomaterials

doi: 10.1093/rb/rbab019

Scatterplots for identification of rat BMSCs via flow cytometry. The expressions of ( a ) CD44, ( b ) CD90 and CD11b, ( c ) CD29 and CD45 were detected in 92.6, 92.8% and 9, 99.6 and 3.9% of the cells, respectively. ( d ) The histogram of cell number. Columns represent mean values and error bars represent SD
Figure Legend Snippet: Scatterplots for identification of rat BMSCs via flow cytometry. The expressions of ( a ) CD44, ( b ) CD90 and CD11b, ( c ) CD29 and CD45 were detected in 92.6, 92.8% and 9, 99.6 and 3.9% of the cells, respectively. ( d ) The histogram of cell number. Columns represent mean values and error bars represent SD

Techniques Used: Flow Cytometry

36) Product Images from "TLR4 Modulates Senescence and Paracrine Action in Placental Mesenchymal Stem Cells via Inhibiting Hedgehog Signaling Pathway in Preeclampsia"

Article Title: TLR4 Modulates Senescence and Paracrine Action in Placental Mesenchymal Stem Cells via Inhibiting Hedgehog Signaling Pathway in Preeclampsia

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2022/7202837

SAG reversed the senescence degree of the placentas and PMSCs in the LPS-induced rats. (a) Western blot analysis of TLR4, SMO, and Gli1 protein expression and quantitative analysis of TLR4, SMO, and Gli1 in different groups of placentas. (b) Western blot analysis and densitometric quantification of p16 and p53 protein expressions in different groups of placentas. (c) SA- β -gal staining of the placentas of different groups of rats. (d) Morphology of rat PMSCs. Small fibroblast- like MSC colonies were detected by an inverted microscope. (e) Successfully differentiated PMSCs of rats were stained with Alizarin Red for osteogenic differentiation, Oil Red O for adipocytes, and alcian blue, in which red calcium nodules, orange lipid droplets, and blue cartilage could be observed. (f) PMSCs of rats expressed CD90 but not CD45. (g) Western blot analysis of TLR4, SMO, and Gli1 protein expression and quantitative analysis of TLR4, SMO, and Gli1 in different groups of PMSCs. (h) Western blot analysis and densitometric quantification of p16 and p53 protein expressions in different groups of placentas. (i) SA- β -gal staining of different groups of rat PMSCs and the average ratio of SA- β -Gal-positive cells in different groups of PMSCs. (j) ELISA analysis of MMP9 concentrations of cell culture medium in different groups of PMSCs. (k) ELISA analysis of sFlt-1 concentrations of cell culture medium in different groups of PMSCs. Scale bar: 200 μ m. Data are presented as the mean ± SD. ∗ P
Figure Legend Snippet: SAG reversed the senescence degree of the placentas and PMSCs in the LPS-induced rats. (a) Western blot analysis of TLR4, SMO, and Gli1 protein expression and quantitative analysis of TLR4, SMO, and Gli1 in different groups of placentas. (b) Western blot analysis and densitometric quantification of p16 and p53 protein expressions in different groups of placentas. (c) SA- β -gal staining of the placentas of different groups of rats. (d) Morphology of rat PMSCs. Small fibroblast- like MSC colonies were detected by an inverted microscope. (e) Successfully differentiated PMSCs of rats were stained with Alizarin Red for osteogenic differentiation, Oil Red O for adipocytes, and alcian blue, in which red calcium nodules, orange lipid droplets, and blue cartilage could be observed. (f) PMSCs of rats expressed CD90 but not CD45. (g) Western blot analysis of TLR4, SMO, and Gli1 protein expression and quantitative analysis of TLR4, SMO, and Gli1 in different groups of PMSCs. (h) Western blot analysis and densitometric quantification of p16 and p53 protein expressions in different groups of placentas. (i) SA- β -gal staining of different groups of rat PMSCs and the average ratio of SA- β -Gal-positive cells in different groups of PMSCs. (j) ELISA analysis of MMP9 concentrations of cell culture medium in different groups of PMSCs. (k) ELISA analysis of sFlt-1 concentrations of cell culture medium in different groups of PMSCs. Scale bar: 200 μ m. Data are presented as the mean ± SD. ∗ P

Techniques Used: Western Blot, Expressing, Staining, Inverted Microscopy, Enzyme-linked Immunosorbent Assay, Cell Culture

37) Product Images from "B1a cells protect against Schistosoma japonicum–induced liver inflammation and fibrosis by controlling monocyte infiltration"

Article Title: B1a cells protect against Schistosoma japonicum–induced liver inflammation and fibrosis by controlling monocyte infiltration

Journal: bioRxiv

doi: 10.1101/420083

Ly6C hi MoMFs are significantly increased in μMT mice. Flow cytometric analysis of macrophage subsets in WT mice and μMT mice after S. japonicum infection. ( A ) Representative fluorescence-activated cell sorting plots are shown for the indicated times after S. japonicum infection. Upper panels are pre-gated on CD45 + and live cells. Bottom panels are pre-gated on CD45 + CD11bhiF4/80lo cells. ( B ) Graphical summary showing percentage of Kupffer cell (KCs) (CD11bloF4/80hi), Ly6C hi MoMF (CD11bhiF4/80loLy6C hi ), and Ly6C lo MoMF (CD11bhiF4/80loLy6C lo ) subsets out of total hepatic macrophags. (C) Absolute numbers of KCs, Ly6C hi MoMFs, and Ly6C lo MoMFs in WT mice and μMT mice. Data represent mean ± SD; n = 8–10 per time point from three independent experiments. * p
Figure Legend Snippet: Ly6C hi MoMFs are significantly increased in μMT mice. Flow cytometric analysis of macrophage subsets in WT mice and μMT mice after S. japonicum infection. ( A ) Representative fluorescence-activated cell sorting plots are shown for the indicated times after S. japonicum infection. Upper panels are pre-gated on CD45 + and live cells. Bottom panels are pre-gated on CD45 + CD11bhiF4/80lo cells. ( B ) Graphical summary showing percentage of Kupffer cell (KCs) (CD11bloF4/80hi), Ly6C hi MoMF (CD11bhiF4/80loLy6C hi ), and Ly6C lo MoMF (CD11bhiF4/80loLy6C lo ) subsets out of total hepatic macrophags. (C) Absolute numbers of KCs, Ly6C hi MoMFs, and Ly6C lo MoMFs in WT mice and μMT mice. Data represent mean ± SD; n = 8–10 per time point from three independent experiments. * p

Techniques Used: Mouse Assay, Infection, Fluorescence, FACS

B cell deficiency results in increased number of macrophages. The infiltration of hepatic leukocytes (CD45 + ), macrophages (CD11b + F4/80 + ), neutrophils (CD11b + Ly6G + ), T cells (CD3 + NK1.1 − ), NK cells (CD3−NK1.1 + ), and NKT cells (CD3 + NK1.1 + ) after infection were quantified by flow cytometric analysis. Controls (Ctrl) were uninfected mice. Data represent mean ± SD; n = 3–5 per time point from three independent experiment. * p
Figure Legend Snippet: B cell deficiency results in increased number of macrophages. The infiltration of hepatic leukocytes (CD45 + ), macrophages (CD11b + F4/80 + ), neutrophils (CD11b + Ly6G + ), T cells (CD3 + NK1.1 − ), NK cells (CD3−NK1.1 + ), and NKT cells (CD3 + NK1.1 + ) after infection were quantified by flow cytometric analysis. Controls (Ctrl) were uninfected mice. Data represent mean ± SD; n = 3–5 per time point from three independent experiment. * p

Techniques Used: Infection, Mouse Assay

38) Product Images from "Single-cell RNA sequencing reveals a strong connection between Gadd45g upregulation and oncolytic HSV infection in tumor tissue"

Article Title: Single-cell RNA sequencing reveals a strong connection between Gadd45g upregulation and oncolytic HSV infection in tumor tissue

Journal: Molecular Therapy Oncolytics

doi: 10.1016/j.omto.2021.10.006

scRNA-seq experiment and data analysis (A) Tumors were explanted from three mice in each group at 48 h after receiving the FusOn-H3 treatment. Tumors were pooled, digested, and dissociated into single cells, which were subsequently sorted into CD45 − and CD45 + populations and then mixed at a 3:1 ratio for scRNA-seq using a 10× Genomics pipeline. (B and C) Aggregated UMAP of all sequenced cells, classified into immune cells (CD45 + / Ptprc ) and tumor cells ( HER2 + ) based on CD45/ Ptprc (B) and HER2 (C) expression. (D) Aggregated UMAP plot showing all sequenced cells from both H7 and H7-HER2 groups, classified into leukocytes, non-infected tumor cells, and infected cells based on viral transcript expression.
Figure Legend Snippet: scRNA-seq experiment and data analysis (A) Tumors were explanted from three mice in each group at 48 h after receiving the FusOn-H3 treatment. Tumors were pooled, digested, and dissociated into single cells, which were subsequently sorted into CD45 − and CD45 + populations and then mixed at a 3:1 ratio for scRNA-seq using a 10× Genomics pipeline. (B and C) Aggregated UMAP of all sequenced cells, classified into immune cells (CD45 + / Ptprc ) and tumor cells ( HER2 + ) based on CD45/ Ptprc (B) and HER2 (C) expression. (D) Aggregated UMAP plot showing all sequenced cells from both H7 and H7-HER2 groups, classified into leukocytes, non-infected tumor cells, and infected cells based on viral transcript expression.

Techniques Used: Mouse Assay, Expressing, Infection

39) Product Images from "Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution"

Article Title: Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2015.00165

Expression of CD markers in freshly isolated and expanded (passage 1) Sk-Cs. (A) Freshly isolated total cells were positive for 6 out of 11 markers, and predominant markers were CD29 and CD34; thus, these were used for subsequent cell fractionation. However, expression of these markers markedly changed after culture of both Sk-DN/29 + and Sk-34/29 +/− cells, but importantly, the characteristics of both cells were similar after expansion. (B) Actual sorting for the exclusion of CD45 + cells, as hematopoietic cells. (C) Basic sorting pattern of 3 cell fractions, as Sk-DN/29 + , Sk-34/29 + and Sk-34/29 − , which was consistently used throughout the present study. Data were obtained from 21 subjects/32 samples (Male n = 19, age 21–79; Female n = 2, age 17–69, from abdominal muscle n = 16, leg muscles n = 5).
Figure Legend Snippet: Expression of CD markers in freshly isolated and expanded (passage 1) Sk-Cs. (A) Freshly isolated total cells were positive for 6 out of 11 markers, and predominant markers were CD29 and CD34; thus, these were used for subsequent cell fractionation. However, expression of these markers markedly changed after culture of both Sk-DN/29 + and Sk-34/29 +/− cells, but importantly, the characteristics of both cells were similar after expansion. (B) Actual sorting for the exclusion of CD45 + cells, as hematopoietic cells. (C) Basic sorting pattern of 3 cell fractions, as Sk-DN/29 + , Sk-34/29 + and Sk-34/29 − , which was consistently used throughout the present study. Data were obtained from 21 subjects/32 samples (Male n = 19, age 21–79; Female n = 2, age 17–69, from abdominal muscle n = 16, leg muscles n = 5).

Techniques Used: Expressing, Isolation, Cell Fractionation

40) Product Images from "Re-evaluating Microglia Expression Profiles Using RiboTag and Cell Isolation Strategies"

Article Title: Re-evaluating Microglia Expression Profiles Using RiboTag and Cell Isolation Strategies

Journal: Nature immunology

doi: 10.1038/s41590-018-0110-6

RiboTag analysis reveals that Cx3cr1 Cre mice but not Cx3cr1 CreER animals display rearrangements in neurons. (A) Scheme of Cx3cr1 Cre and Cx3cr1 CreER systems. (B) Scheme describing the immuno-precipitation protocol, including brain homogenization, centrifugation to remove cell debris and incubation with magnetic beads and relevant antibodies. (C) Heat maps of RNAseq data comparing IPs obtained from brains of Cx3cr1 Cre :Rpl22 HA and Cx3cr1 CreER :Rpl22 HA mice, represented by lists of genes of microglia (115), neurons (97), astrocytes (95) and oligodendrocytes (98) showing enrichment and de-enrichment of mRNAs of specific cell types in the different samples. Reference data sets 9 . Each column represents an individual mouse, n=2 for Cx3cr1 CreER no TAM, n=3 for Cx3cr1 Cre and Cx3cr1 CreER with TAM. (D) Microscopic analysis of cortex brain sections from Cx3cr1 Cre :R26-YFP mice (left panel) and Cx3cr1 CreER :R26-YFP mice (TAM treated (right panel) or untreated controls (middle panel)), stained for IBA-1, YFP and DAPI, showing neuronal expression of YFP in Cx3cr1 Cre brains and microglia-restricted YFP expression in Cx3cr1 CreER brains. The animals analyzed are F1 offspring of the intercross of homozygote Cx3cr1 CreER or Cx3cr1 Cre animals and homozygote R26-YFP mice. Representative of 2 independent experiments. (E) Immuno-fluorescent staining of tissue sections of Cx3cr1 Cre :Rpl22 HA (left) and TAM-treated Cx3cr1 CreER :Rpl22 HA (right) mice, stained for IBA1, HA and Hoechst, showing neuronal expression of HA in Cx3cr1 Cre cortex, and microglia-restricted HA expression in Cx3cr1 CreER spinal cord. Scale bars: 200µm (left), 50µm (right) Representative of 2 independent experiments. (F) Flow cytometry analysis showing HA staining in microglia (CD11b + CD45 int , gated on Ly6C/G – DAPI – ) of Rpl22 HA TAM-treated mice (black line), Cx3cr1 CreER :Rpl22 HA mice, untreated (blue line) and TAM-treated (red line). Shadowed histogram represents isotype (IgG) control. Representative data of 3 repeats.
Figure Legend Snippet: RiboTag analysis reveals that Cx3cr1 Cre mice but not Cx3cr1 CreER animals display rearrangements in neurons. (A) Scheme of Cx3cr1 Cre and Cx3cr1 CreER systems. (B) Scheme describing the immuno-precipitation protocol, including brain homogenization, centrifugation to remove cell debris and incubation with magnetic beads and relevant antibodies. (C) Heat maps of RNAseq data comparing IPs obtained from brains of Cx3cr1 Cre :Rpl22 HA and Cx3cr1 CreER :Rpl22 HA mice, represented by lists of genes of microglia (115), neurons (97), astrocytes (95) and oligodendrocytes (98) showing enrichment and de-enrichment of mRNAs of specific cell types in the different samples. Reference data sets 9 . Each column represents an individual mouse, n=2 for Cx3cr1 CreER no TAM, n=3 for Cx3cr1 Cre and Cx3cr1 CreER with TAM. (D) Microscopic analysis of cortex brain sections from Cx3cr1 Cre :R26-YFP mice (left panel) and Cx3cr1 CreER :R26-YFP mice (TAM treated (right panel) or untreated controls (middle panel)), stained for IBA-1, YFP and DAPI, showing neuronal expression of YFP in Cx3cr1 Cre brains and microglia-restricted YFP expression in Cx3cr1 CreER brains. The animals analyzed are F1 offspring of the intercross of homozygote Cx3cr1 CreER or Cx3cr1 Cre animals and homozygote R26-YFP mice. Representative of 2 independent experiments. (E) Immuno-fluorescent staining of tissue sections of Cx3cr1 Cre :Rpl22 HA (left) and TAM-treated Cx3cr1 CreER :Rpl22 HA (right) mice, stained for IBA1, HA and Hoechst, showing neuronal expression of HA in Cx3cr1 Cre cortex, and microglia-restricted HA expression in Cx3cr1 CreER spinal cord. Scale bars: 200µm (left), 50µm (right) Representative of 2 independent experiments. (F) Flow cytometry analysis showing HA staining in microglia (CD11b + CD45 int , gated on Ly6C/G – DAPI – ) of Rpl22 HA TAM-treated mice (black line), Cx3cr1 CreER :Rpl22 HA mice, untreated (blue line) and TAM-treated (red line). Shadowed histogram represents isotype (IgG) control. Representative data of 3 repeats.

Techniques Used: Mouse Assay, Immunoprecipitation, Homogenization, Centrifugation, Incubation, Magnetic Beads, Staining, Expressing, Flow Cytometry, Cytometry

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    BioLegend anti cd4
    ADP-ribosylation of Trm during cell preparation induces cell death upon 37°C incubation. (A) NAD + is released during cell preparation and serves as substrate for ARTC2.2 to ADP-ribosylate P2X7 at R125, even if cells are prepared at 4°C. ADP-ribosylation-mediated gating of P2X7 occurs when cells are brought back to 37°C, inducing Ca 2+ influx and ultimately cell death. ADP-ribosylation of P2X7 during cell preparation and subsequent activation of P2X7 at 37°C can be prevented by injection of the ARTC2.2-blocking nanobody s+16a 30 min prior to sacrificing the mice. (B) CD8 + and <t>CD4</t> + Trm were isolated via FACS from the liver of naïve WT, ARTC2ko, and P2X7ko mice. Cells were incubated at 37°C for 2 h and propidium iodide (PI) uptake was measured by flow cytometry as marker for cell death. (C) Isolated CD8 + and CD4 + Trm from the liver of naïve WT mice were cultured in the presence or absence of eFluor 670 -labeled feeder cells in a ration of 1:20. Cells were incubated at 37°C for 2 h and PI uptake by Trm was measured by flow cytometry as marker for cell death. (D) CD8 + and CD4 + Trm and effector memory T cells (Tem) were isolated via FACS from the liver of Lm infected mice 7 weeks after infection. One group of mice was treated with s+16a prior to organ harvesting and the second group was left untreated as control. Cells were incubated at 37°C for 2 h and PI uptake was measured by flow cytometry as marker for cell death. The shown data represent results from two independently performed experiments.
    Anti Cd4, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend cd69
    Ability of DC produced in co-cultures to induce T cell responses. Co-cultures were established by overlay of Lin − BM from Act-mOVA mice above 5G3 stroma and non-adherent cells collected after 21 days. Cells were stained for CD11b, CD11c and MHC-II expression and subsets of L-DC and cDC-like cells sorted and then incubated with CD8 + T cells purified from OT-I TCR-tg mice specific for OVA 257–264 /H-2K b or OT-II TCR-tg mice specific for OVA 323–339 /H-2IA b at APC:T cell ratios of 1:10, 1:100 and 1:1000. Controls included T cells only with no APC. Control APC included CD11c + DC from spleen. ( a ) Cells for analysis of OT-I responses were collected from cultures and gated as live (PI − ) Thy1.2 + Vα2 + CD8 + T cells. T cell activation was measured at 24 h in terms of % cells expressing <t>CD69.</t> T cell proliferation was measured after 4 days in terms of a reduction in CFSE staining. Data represent mean ± S.E. of three replicate co-cultures. L-DC gave statistically greater T cell activation across all three APC:T cell ratios, while statistically greater T cell proliferation was noted only at an APC:T cell ratio of 10:1 ( p ≤ 0.05). ( b ) Cells for analysis of OT-II responses were collected from cultures and T cells gated as live (PI − ) Thy1.2 + Vα2 + CD4 + cells. T cell activation was measured at 24 h as % cells expressing CD69. T cell proliferation was measured at 4 days in terms of reduction in CFSE staining. Production of regulatory T cells was assessed through intracellular Foxp3 expression in CD4 + T cells analysed after 4 days by flow cytometry
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    Silencing CIITA attenuates α-syn-mediated T cell and monocyte entry. a Representative flow cytometry plots display gating of lymphoid cells (left, representative percent of displayed population is given on plot) followed by <t>CD4</t> and CD8+ T cells (from the CD45 hi CD11b − population, right, mean raw cell number ± SEM is shown on plot) in the ventral midbrains of mice bilaterally injected with AAV2-SYN + LGFP, AAV2-SYN + LVA, or AAV2-SYN + LVE at 4 weeks post-transduction. b Quantification of absolute numbers of CD4 and CD8 T cells from a . c Representative flow cytometry plots showing gating on LY6C hi monocytes (from the CD45 hi CD11b + population, mean raw cell number ± SEM is shown on plot) in the ventral midbrains of mice bilaterally injected with AAV2-SYN + LGFP, AAV2-SYN + LVA, or AAV2-SYN + LVE at 4 weeks post-transduction. d Quantification of the LY6C hi monocytes from c . For a – d , a total of 24 mice were used—each group consisted of 4 samples with 2 ventral midbrains pooled per sample. Equal numbers of male and female mice were used in these experiments. The mean +/− SEM of samples in each group are plotted. A one-way ANOVA with Tukey’s post hoc test was used for statistical analysis. * p
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    ADP-ribosylation of Trm during cell preparation induces cell death upon 37°C incubation. (A) NAD + is released during cell preparation and serves as substrate for ARTC2.2 to ADP-ribosylate P2X7 at R125, even if cells are prepared at 4°C. ADP-ribosylation-mediated gating of P2X7 occurs when cells are brought back to 37°C, inducing Ca 2+ influx and ultimately cell death. ADP-ribosylation of P2X7 during cell preparation and subsequent activation of P2X7 at 37°C can be prevented by injection of the ARTC2.2-blocking nanobody s+16a 30 min prior to sacrificing the mice. (B) CD8 + and CD4 + Trm were isolated via FACS from the liver of naïve WT, ARTC2ko, and P2X7ko mice. Cells were incubated at 37°C for 2 h and propidium iodide (PI) uptake was measured by flow cytometry as marker for cell death. (C) Isolated CD8 + and CD4 + Trm from the liver of naïve WT mice were cultured in the presence or absence of eFluor 670 -labeled feeder cells in a ration of 1:20. Cells were incubated at 37°C for 2 h and PI uptake by Trm was measured by flow cytometry as marker for cell death. (D) CD8 + and CD4 + Trm and effector memory T cells (Tem) were isolated via FACS from the liver of Lm infected mice 7 weeks after infection. One group of mice was treated with s+16a prior to organ harvesting and the second group was left untreated as control. Cells were incubated at 37°C for 2 h and PI uptake was measured by flow cytometry as marker for cell death. The shown data represent results from two independently performed experiments.

    Journal: Frontiers in Immunology

    Article Title: In Vivo Blockade of Murine ARTC2.2 During Cell Preparation Preserves the Vitality and Function of Liver Tissue-Resident Memory T Cells

    doi: 10.3389/fimmu.2018.01580

    Figure Lengend Snippet: ADP-ribosylation of Trm during cell preparation induces cell death upon 37°C incubation. (A) NAD + is released during cell preparation and serves as substrate for ARTC2.2 to ADP-ribosylate P2X7 at R125, even if cells are prepared at 4°C. ADP-ribosylation-mediated gating of P2X7 occurs when cells are brought back to 37°C, inducing Ca 2+ influx and ultimately cell death. ADP-ribosylation of P2X7 during cell preparation and subsequent activation of P2X7 at 37°C can be prevented by injection of the ARTC2.2-blocking nanobody s+16a 30 min prior to sacrificing the mice. (B) CD8 + and CD4 + Trm were isolated via FACS from the liver of naïve WT, ARTC2ko, and P2X7ko mice. Cells were incubated at 37°C for 2 h and propidium iodide (PI) uptake was measured by flow cytometry as marker for cell death. (C) Isolated CD8 + and CD4 + Trm from the liver of naïve WT mice were cultured in the presence or absence of eFluor 670 -labeled feeder cells in a ration of 1:20. Cells were incubated at 37°C for 2 h and PI uptake by Trm was measured by flow cytometry as marker for cell death. (D) CD8 + and CD4 + Trm and effector memory T cells (Tem) were isolated via FACS from the liver of Lm infected mice 7 weeks after infection. One group of mice was treated with s+16a prior to organ harvesting and the second group was left untreated as control. Cells were incubated at 37°C for 2 h and PI uptake was measured by flow cytometry as marker for cell death. The shown data represent results from two independently performed experiments.

    Article Snippet: Antibodies and Flow Cytometry The following antibodies were used for flow cytometric analyses: anti-ARTC2.2 (clone Nika109; UKE), anti-CD3 (clone 145-2C11, BioLegend), anti-CD4 (clone RM4–5, BioLegend), anti-CD8 (clone 53-6.7, BioLegend), anti-CD45 (clone30-F11, BioLegend), anti-CD69 (clone H1.2F3, BioLegend), anti-KLRG1 (clone 2F1/KLRG1, BioLegend), and anti-P2X7 (clone RH23A44, UKE).

    Techniques: Incubation, IF-cells, Activation Assay, Injection, Blocking Assay, Mouse Assay, Isolation, FACS, Flow Cytometry, Cytometry, Marker, Cell Culture, Labeling, Transmission Electron Microscopy, Infection

    Liver Trm co-express high levels of ARTC2.2 and P2X7. (A) C57BL/6 mice were infected i.v. with 2 × 10 4 Listeria monocytogenes (Lm). Seven weeks after infection, mice were treated with perCP-labeled anti-CD45 3 min before sacrificing to label vascular leukocytes. The liver of treated mice was harvested for Trm analyses. (B) Gating strategy: within the CD3 + CD1d tet− T cell pool Trm were identified as CD8 + CD69 + KLRG1 − or CD4 + CD69 + KLRG1 − and effector memory T cells (Tem) as CD8 + CD69 − KLRG1 + or CD4 + CD69 − KLRG1 + ; double negative (DN) marks CD8 + or CD4 + T cells that were CD69 − KLRG1 − . (C) In vivo anti-CD45 labeling (CD45 blood ) of Trm, Tem, and DN in relation to ex vivo anti-CD45 labeling (CD45 all ). (D) Frequencies of CD8 + and CD4 + Trm and Tem in the liver of naïve and Lm infected mice. Two groups were compared using Student’s t -test ( n = 8–16) with * p

    Journal: Frontiers in Immunology

    Article Title: In Vivo Blockade of Murine ARTC2.2 During Cell Preparation Preserves the Vitality and Function of Liver Tissue-Resident Memory T Cells

    doi: 10.3389/fimmu.2018.01580

    Figure Lengend Snippet: Liver Trm co-express high levels of ARTC2.2 and P2X7. (A) C57BL/6 mice were infected i.v. with 2 × 10 4 Listeria monocytogenes (Lm). Seven weeks after infection, mice were treated with perCP-labeled anti-CD45 3 min before sacrificing to label vascular leukocytes. The liver of treated mice was harvested for Trm analyses. (B) Gating strategy: within the CD3 + CD1d tet− T cell pool Trm were identified as CD8 + CD69 + KLRG1 − or CD4 + CD69 + KLRG1 − and effector memory T cells (Tem) as CD8 + CD69 − KLRG1 + or CD4 + CD69 − KLRG1 + ; double negative (DN) marks CD8 + or CD4 + T cells that were CD69 − KLRG1 − . (C) In vivo anti-CD45 labeling (CD45 blood ) of Trm, Tem, and DN in relation to ex vivo anti-CD45 labeling (CD45 all ). (D) Frequencies of CD8 + and CD4 + Trm and Tem in the liver of naïve and Lm infected mice. Two groups were compared using Student’s t -test ( n = 8–16) with * p

    Article Snippet: Antibodies and Flow Cytometry The following antibodies were used for flow cytometric analyses: anti-ARTC2.2 (clone Nika109; UKE), anti-CD3 (clone 145-2C11, BioLegend), anti-CD4 (clone RM4–5, BioLegend), anti-CD8 (clone 53-6.7, BioLegend), anti-CD45 (clone30-F11, BioLegend), anti-CD69 (clone H1.2F3, BioLegend), anti-KLRG1 (clone 2F1/KLRG1, BioLegend), and anti-P2X7 (clone RH23A44, UKE).

    Techniques: Mouse Assay, Infection, Labeling, Transmission Electron Microscopy, In Vivo, Ex Vivo

    In vitro cytokine expression profile of PMA/ionomycin stimulated liver Trm. (A) CD8 + (1 × 10 4 cells) and CD4 + (2 × 10 4 cells) Trm from the liver of Lm infected mice were FACS sorted 7 weeks after infection. One group of mice was treated with s+16a prior to organ harvesting and the second group was left untreated as control. Cells were stimulated with PMA/ionomycin for 20 h and T cell cytokines [IFNγ, TNFα, interleukin (IL)-2, IL-4, IL-21, IL-22, IL-17A, IL-17F, IL-10, IL-9, IL-5, and IL-13] were measured in the supernatants using a bead-based 13-plex assay. (B) The concentration of cytokines in the supernatants of stimulated CD4 + and CD8 + Trm from s+16a-treated and control mice ( n = 3) was quantified.

    Journal: Frontiers in Immunology

    Article Title: In Vivo Blockade of Murine ARTC2.2 During Cell Preparation Preserves the Vitality and Function of Liver Tissue-Resident Memory T Cells

    doi: 10.3389/fimmu.2018.01580

    Figure Lengend Snippet: In vitro cytokine expression profile of PMA/ionomycin stimulated liver Trm. (A) CD8 + (1 × 10 4 cells) and CD4 + (2 × 10 4 cells) Trm from the liver of Lm infected mice were FACS sorted 7 weeks after infection. One group of mice was treated with s+16a prior to organ harvesting and the second group was left untreated as control. Cells were stimulated with PMA/ionomycin for 20 h and T cell cytokines [IFNγ, TNFα, interleukin (IL)-2, IL-4, IL-21, IL-22, IL-17A, IL-17F, IL-10, IL-9, IL-5, and IL-13] were measured in the supernatants using a bead-based 13-plex assay. (B) The concentration of cytokines in the supernatants of stimulated CD4 + and CD8 + Trm from s+16a-treated and control mice ( n = 3) was quantified.

    Article Snippet: Antibodies and Flow Cytometry The following antibodies were used for flow cytometric analyses: anti-ARTC2.2 (clone Nika109; UKE), anti-CD3 (clone 145-2C11, BioLegend), anti-CD4 (clone RM4–5, BioLegend), anti-CD8 (clone 53-6.7, BioLegend), anti-CD45 (clone30-F11, BioLegend), anti-CD69 (clone H1.2F3, BioLegend), anti-KLRG1 (clone 2F1/KLRG1, BioLegend), and anti-P2X7 (clone RH23A44, UKE).

    Techniques: In Vitro, Expressing, Infection, Mouse Assay, FACS, Plex Assay, Concentration Assay

    Tfr cells regulate antibody memory responses a) Schematic of Tfr-deletion to assess memory responses. Tfr-DTR or control mice were immunized with NP-OVA in MF59 and DT was administered on days 5,7 and 9 to delete Tfr cells prior to GC formation. Mice received a boost of NP-OVA without adjuvant at d30. Mice were harvested on d38. b) Analysis of NP-specific IgG levels before and after NP-OVA boost as in (a). c) Quantification of the NP2/NP16 ratio in experiments as in (a). d) Quantification of GC B cells (gated as CD19 + GL7 + FAS + ) from dLN of mice at d38 as in (a). e) Quantification of Tfh (gated as CD4 + ICOS + CXCR5 + FoxP3-CD19-) cells at d38 as in (a). f) Quantification of Ki67 expression in Tfh cells gated as in (e). Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data represent combined data from 3 independent experiments.

    Journal: Nature immunology

    Article Title: Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses

    doi: 10.1038/s41590-019-0472-4

    Figure Lengend Snippet: Tfr cells regulate antibody memory responses a) Schematic of Tfr-deletion to assess memory responses. Tfr-DTR or control mice were immunized with NP-OVA in MF59 and DT was administered on days 5,7 and 9 to delete Tfr cells prior to GC formation. Mice received a boost of NP-OVA without adjuvant at d30. Mice were harvested on d38. b) Analysis of NP-specific IgG levels before and after NP-OVA boost as in (a). c) Quantification of the NP2/NP16 ratio in experiments as in (a). d) Quantification of GC B cells (gated as CD19 + GL7 + FAS + ) from dLN of mice at d38 as in (a). e) Quantification of Tfh (gated as CD4 + ICOS + CXCR5 + FoxP3-CD19-) cells at d38 as in (a). f) Quantification of Ki67 expression in Tfh cells gated as in (e). Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data represent combined data from 3 independent experiments.

    Article Snippet: The following antibodies were used for surface staining at 4o C for 30 minutes: anti-CD4 (Biolegend, 1:200, RM4–5), anti-ICOS (Biolegend,1:200, 15F9), anti-CD19 (Biolegend,1:200, 6D5), anti-PD-1 (1:200, RMP1–30), anti-CXCR5 biotin (BD Biosciences,1:100, 2G8), anti-GL7 (BD Biosciences,1:200, GL-7), anti-HB-EGF/DTR (RandD Systems, 1:200, AF-259-NA), anti-CD38 (Biolegend, 1:200, 90), anti-CD138 (Biolegend, 1:200, 281–2), anti-IA (Biolegend,1:200, M5/114.15.2), anti-SiglecF (BD Biosciences, 1:200, E50–2440), anti-CD8a (Biolegend, 1:200, 53–6.7), anti-CD11c (Biolegend, 1:200, N418), anti-CD11b (Biolegend, 1:200, M1/70).

    Techniques: Mouse Assay, Expressing, Two Tailed Test

    Tfr cells Potently Regulate Early Geminal Center Formation a) Quantification of Tfr (gated as CD4 + ICOS + CXCR5 + FoxP3 + CD19 − ) and Tfh (gated as CD4 + ICOS + CXCR5 + FoxP3 − CD19 − ) cells from dLNs of Tfr-DTR ( Foxp3 Cre Cxcr5 LoxStopLoxDTR/WT ) or Control ( Foxp3 Cre Cxcr5 WT/WT ) mice 21 days after immunization. Diphtheria toxin (DT) was administered on days 5,7 and 9 to delete Tfr cells before GC initiation. b) Quantification of GC B cells (gated as CD19 + GL7 + FAS + ) and naive B cells (gated as CD38 + IgG1 − ) from dLNs at d21 after immunization as in (a). c) Quantification of plasma cells (gated as CD138 + ), class switched B cells (gated as CD19 + IgG1 + CD38-), and memory-like B cells (gated as CD19 + IgG1 + CD38 + ) at d21 after immunization as in (a). d) Glut1 expression on B cells from mice as in (a). Representative histogram (left) and quantification (right). e) Quantification of total IgG (far left), NP-specific IgG (middle left), total IgE (middle right) and total IgA (far right) analyzed from serum of mice as in (a). f) Quantification of Tfr (gated as CD4 + ICOS + CXCR5 + FoxP3 + CD19 − ) and Tfh (gated as CD4 + ICOS + CXCR5 + FoxP3 − CD19 − ) cells from dLNs of Tfr-DTR ( Foxp3 Cre Cxcr5 LoxStopLoxDTR/WT ) or Control ( Foxp3 Cre Cxcr5 WT/WT ) mice at d21 after immunization. Diphtheria toxin (DT) was administered on days 10, 12 and 14 to delete Tfr cells after GC formation. g) Quantification of GC B cells (gated as CD19 + GL7 + FAS + ) and plasma cells (CD138 + ) from dLNs at d21 after immunization as in (e). h) Quantification of total IgG (left) and NP-specific IgG (left). Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data are combined results from 4 (a-c,e) or 3 (f-h) independent experiments, or are from an individual experiment which is representative of two independent experiments (d).

    Journal: Nature immunology

    Article Title: Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses

    doi: 10.1038/s41590-019-0472-4

    Figure Lengend Snippet: Tfr cells Potently Regulate Early Geminal Center Formation a) Quantification of Tfr (gated as CD4 + ICOS + CXCR5 + FoxP3 + CD19 − ) and Tfh (gated as CD4 + ICOS + CXCR5 + FoxP3 − CD19 − ) cells from dLNs of Tfr-DTR ( Foxp3 Cre Cxcr5 LoxStopLoxDTR/WT ) or Control ( Foxp3 Cre Cxcr5 WT/WT ) mice 21 days after immunization. Diphtheria toxin (DT) was administered on days 5,7 and 9 to delete Tfr cells before GC initiation. b) Quantification of GC B cells (gated as CD19 + GL7 + FAS + ) and naive B cells (gated as CD38 + IgG1 − ) from dLNs at d21 after immunization as in (a). c) Quantification of plasma cells (gated as CD138 + ), class switched B cells (gated as CD19 + IgG1 + CD38-), and memory-like B cells (gated as CD19 + IgG1 + CD38 + ) at d21 after immunization as in (a). d) Glut1 expression on B cells from mice as in (a). Representative histogram (left) and quantification (right). e) Quantification of total IgG (far left), NP-specific IgG (middle left), total IgE (middle right) and total IgA (far right) analyzed from serum of mice as in (a). f) Quantification of Tfr (gated as CD4 + ICOS + CXCR5 + FoxP3 + CD19 − ) and Tfh (gated as CD4 + ICOS + CXCR5 + FoxP3 − CD19 − ) cells from dLNs of Tfr-DTR ( Foxp3 Cre Cxcr5 LoxStopLoxDTR/WT ) or Control ( Foxp3 Cre Cxcr5 WT/WT ) mice at d21 after immunization. Diphtheria toxin (DT) was administered on days 10, 12 and 14 to delete Tfr cells after GC formation. g) Quantification of GC B cells (gated as CD19 + GL7 + FAS + ) and plasma cells (CD138 + ) from dLNs at d21 after immunization as in (e). h) Quantification of total IgG (left) and NP-specific IgG (left). Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data are combined results from 4 (a-c,e) or 3 (f-h) independent experiments, or are from an individual experiment which is representative of two independent experiments (d).

    Article Snippet: The following antibodies were used for surface staining at 4o C for 30 minutes: anti-CD4 (Biolegend, 1:200, RM4–5), anti-ICOS (Biolegend,1:200, 15F9), anti-CD19 (Biolegend,1:200, 6D5), anti-PD-1 (1:200, RMP1–30), anti-CXCR5 biotin (BD Biosciences,1:100, 2G8), anti-GL7 (BD Biosciences,1:200, GL-7), anti-HB-EGF/DTR (RandD Systems, 1:200, AF-259-NA), anti-CD38 (Biolegend, 1:200, 90), anti-CD138 (Biolegend, 1:200, 281–2), anti-IA (Biolegend,1:200, M5/114.15.2), anti-SiglecF (BD Biosciences, 1:200, E50–2440), anti-CD8a (Biolegend, 1:200, 53–6.7), anti-CD11c (Biolegend, 1:200, N418), anti-CD11b (Biolegend, 1:200, M1/70).

    Techniques: Mouse Assay, Expressing, Two Tailed Test

    Tfr cells regulate HDM-specific IgE responses in vivo a) Schematic of HDM sensitization and challenge model to induce lung inflammation. Tfr-DTR ( Foxp3 Cre Cxcr5 LoxStopLoxDTR/WT ) or Control ( Foxp3 Cre Cxcr5 WT/WT ) mice received HDM sensitization at day 0 followed by DT administration at days 0,2,4 and 6. Mice were challenged with HDM on days 7–11 and harvested on day 15. b) Analysis of Tfr cells from dLN of HDM challenged mice as in (a). Representative gating (left) and quantification (right) are shown. c) Quantification of Tfh, GC B cells (CD19 + GL7 + FAS + ), total IgE + B cells (CD19 + IgE + ), total IgG1 + B cells (CD19 + IgG1 + ) and total plasma cells (CD138 + ) from dLN of HDM challenged mice as in (a). d) Quantification of IgG1 and IgE expression in GC B cells (CD19 + GL7 + FAS + ). e) Quantification of IgG1 and IgE expression in plasma cells (CD138 + ). f) Quantification of total IgE or HDM-specific IgE from HDM challenged mice as in (a). (n=30, Control; n=22, Tfr-DTR) g) Quantification of Eosinophils (left), CD4 T cells (middle) or CD8 T cells (right) from the BAL fluid of mice as in (a). (n=24, Control; n=17, Tfr-DTR) h) Lung inflammation scores (left) and representative images of H E or PAS staining (right) of lung samples. Scale bars = 500μM. i) Immunofluorescence micrographs of lungs stained for Actin, SiglecF, Gr1 and I-A. Scale bars = 100μM. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test (b-e) or Mann-Whitney (f-h). Data are from individual experiments and are representative of 3 independent experiments (b, c left), are combined data from 4 independent experiments (c right, d-g), or are from one experiment (h-i).

    Journal: Nature immunology

    Article Title: Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses

    doi: 10.1038/s41590-019-0472-4

    Figure Lengend Snippet: Tfr cells regulate HDM-specific IgE responses in vivo a) Schematic of HDM sensitization and challenge model to induce lung inflammation. Tfr-DTR ( Foxp3 Cre Cxcr5 LoxStopLoxDTR/WT ) or Control ( Foxp3 Cre Cxcr5 WT/WT ) mice received HDM sensitization at day 0 followed by DT administration at days 0,2,4 and 6. Mice were challenged with HDM on days 7–11 and harvested on day 15. b) Analysis of Tfr cells from dLN of HDM challenged mice as in (a). Representative gating (left) and quantification (right) are shown. c) Quantification of Tfh, GC B cells (CD19 + GL7 + FAS + ), total IgE + B cells (CD19 + IgE + ), total IgG1 + B cells (CD19 + IgG1 + ) and total plasma cells (CD138 + ) from dLN of HDM challenged mice as in (a). d) Quantification of IgG1 and IgE expression in GC B cells (CD19 + GL7 + FAS + ). e) Quantification of IgG1 and IgE expression in plasma cells (CD138 + ). f) Quantification of total IgE or HDM-specific IgE from HDM challenged mice as in (a). (n=30, Control; n=22, Tfr-DTR) g) Quantification of Eosinophils (left), CD4 T cells (middle) or CD8 T cells (right) from the BAL fluid of mice as in (a). (n=24, Control; n=17, Tfr-DTR) h) Lung inflammation scores (left) and representative images of H E or PAS staining (right) of lung samples. Scale bars = 500μM. i) Immunofluorescence micrographs of lungs stained for Actin, SiglecF, Gr1 and I-A. Scale bars = 100μM. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test (b-e) or Mann-Whitney (f-h). Data are from individual experiments and are representative of 3 independent experiments (b, c left), are combined data from 4 independent experiments (c right, d-g), or are from one experiment (h-i).

    Article Snippet: The following antibodies were used for surface staining at 4o C for 30 minutes: anti-CD4 (Biolegend, 1:200, RM4–5), anti-ICOS (Biolegend,1:200, 15F9), anti-CD19 (Biolegend,1:200, 6D5), anti-PD-1 (1:200, RMP1–30), anti-CXCR5 biotin (BD Biosciences,1:100, 2G8), anti-GL7 (BD Biosciences,1:200, GL-7), anti-HB-EGF/DTR (RandD Systems, 1:200, AF-259-NA), anti-CD38 (Biolegend, 1:200, 90), anti-CD138 (Biolegend, 1:200, 281–2), anti-IA (Biolegend,1:200, M5/114.15.2), anti-SiglecF (BD Biosciences, 1:200, E50–2440), anti-CD8a (Biolegend, 1:200, 53–6.7), anti-CD11c (Biolegend, 1:200, N418), anti-CD11b (Biolegend, 1:200, M1/70).

    Techniques: In Vivo, Mouse Assay, Expressing, Staining, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

    Tfr cells regulate Tfh13-mediated IgE responses in vitro a) Schematic of experimental design for an in vitro HDM suppression assay. Total B, Tfh and Tfr cells were purified from the dLN of mice that received HDM on days 0,2,4 and 6 and added to culture wells along with HDM for 6 days. b) Quantification of total Tfh cells (left) and the percentage of Tfr cells (FoxP3 + of CD4 + IA-CD19- cells) (right) from cultures as in (a). c) Quantification of cytokines in culture supernatants from cultures as in (a). Cytokines listed in red have levels statistically lower in cultures containing Tfr cells compared to cultures without Tfr cells. d) Column graphs of IL5, IL4 and IL13 from data in (c). e) Intracellular cytokine staining of IL-13 in Tfh cells from cultures as in (a). f) Analysis of class switching to IgG1 and IgE in cultures as in (a). Representative gating (left, pregated on CD19 + IA + CD4- cells), IgE + B cell quantification (middle) and IgG1 + B cell quantification (right) are shown. g) Counts of total IgE + (left) and IgG1 + (right) B cells expressed as relative counts from experiments as in (a). h) Expression of GL7 on IgE + B cells from indicated cultures as in (a). i) Expression of IgE on IgE + B cells (left) and IgG1 on IgG1 + B cells are shown from cultures as in (a). j) Levels of IgE (left) and IgG (right) in culture supernatants from cultures as in (a). Anti-IL13 or anti-IL4 were added to indicated wells. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test (b-i, j; right) or One-way ANOVA with Tukey’s correction (j; left). Data are from individual experiments and are representative of 3 independent experiments.

    Journal: Nature immunology

    Article Title: Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses

    doi: 10.1038/s41590-019-0472-4

    Figure Lengend Snippet: Tfr cells regulate Tfh13-mediated IgE responses in vitro a) Schematic of experimental design for an in vitro HDM suppression assay. Total B, Tfh and Tfr cells were purified from the dLN of mice that received HDM on days 0,2,4 and 6 and added to culture wells along with HDM for 6 days. b) Quantification of total Tfh cells (left) and the percentage of Tfr cells (FoxP3 + of CD4 + IA-CD19- cells) (right) from cultures as in (a). c) Quantification of cytokines in culture supernatants from cultures as in (a). Cytokines listed in red have levels statistically lower in cultures containing Tfr cells compared to cultures without Tfr cells. d) Column graphs of IL5, IL4 and IL13 from data in (c). e) Intracellular cytokine staining of IL-13 in Tfh cells from cultures as in (a). f) Analysis of class switching to IgG1 and IgE in cultures as in (a). Representative gating (left, pregated on CD19 + IA + CD4- cells), IgE + B cell quantification (middle) and IgG1 + B cell quantification (right) are shown. g) Counts of total IgE + (left) and IgG1 + (right) B cells expressed as relative counts from experiments as in (a). h) Expression of GL7 on IgE + B cells from indicated cultures as in (a). i) Expression of IgE on IgE + B cells (left) and IgG1 on IgG1 + B cells are shown from cultures as in (a). j) Levels of IgE (left) and IgG (right) in culture supernatants from cultures as in (a). Anti-IL13 or anti-IL4 were added to indicated wells. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test (b-i, j; right) or One-way ANOVA with Tukey’s correction (j; left). Data are from individual experiments and are representative of 3 independent experiments.

    Article Snippet: The following antibodies were used for surface staining at 4o C for 30 minutes: anti-CD4 (Biolegend, 1:200, RM4–5), anti-ICOS (Biolegend,1:200, 15F9), anti-CD19 (Biolegend,1:200, 6D5), anti-PD-1 (1:200, RMP1–30), anti-CXCR5 biotin (BD Biosciences,1:100, 2G8), anti-GL7 (BD Biosciences,1:200, GL-7), anti-HB-EGF/DTR (RandD Systems, 1:200, AF-259-NA), anti-CD38 (Biolegend, 1:200, 90), anti-CD138 (Biolegend, 1:200, 281–2), anti-IA (Biolegend,1:200, M5/114.15.2), anti-SiglecF (BD Biosciences, 1:200, E50–2440), anti-CD8a (Biolegend, 1:200, 53–6.7), anti-CD11c (Biolegend, 1:200, N418), anti-CD11b (Biolegend, 1:200, M1/70).

    Techniques: In Vitro, Suppression Assay, Purification, Mouse Assay, Staining, Expressing, Two Tailed Test

    Ability of DC produced in co-cultures to induce T cell responses. Co-cultures were established by overlay of Lin − BM from Act-mOVA mice above 5G3 stroma and non-adherent cells collected after 21 days. Cells were stained for CD11b, CD11c and MHC-II expression and subsets of L-DC and cDC-like cells sorted and then incubated with CD8 + T cells purified from OT-I TCR-tg mice specific for OVA 257–264 /H-2K b or OT-II TCR-tg mice specific for OVA 323–339 /H-2IA b at APC:T cell ratios of 1:10, 1:100 and 1:1000. Controls included T cells only with no APC. Control APC included CD11c + DC from spleen. ( a ) Cells for analysis of OT-I responses were collected from cultures and gated as live (PI − ) Thy1.2 + Vα2 + CD8 + T cells. T cell activation was measured at 24 h in terms of % cells expressing CD69. T cell proliferation was measured after 4 days in terms of a reduction in CFSE staining. Data represent mean ± S.E. of three replicate co-cultures. L-DC gave statistically greater T cell activation across all three APC:T cell ratios, while statistically greater T cell proliferation was noted only at an APC:T cell ratio of 10:1 ( p ≤ 0.05). ( b ) Cells for analysis of OT-II responses were collected from cultures and T cells gated as live (PI − ) Thy1.2 + Vα2 + CD4 + cells. T cell activation was measured at 24 h as % cells expressing CD69. T cell proliferation was measured at 4 days in terms of reduction in CFSE staining. Production of regulatory T cells was assessed through intracellular Foxp3 expression in CD4 + T cells analysed after 4 days by flow cytometry

    Journal: BMC Immunology

    Article Title: MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis

    doi: 10.1186/s12865-018-0255-y

    Figure Lengend Snippet: Ability of DC produced in co-cultures to induce T cell responses. Co-cultures were established by overlay of Lin − BM from Act-mOVA mice above 5G3 stroma and non-adherent cells collected after 21 days. Cells were stained for CD11b, CD11c and MHC-II expression and subsets of L-DC and cDC-like cells sorted and then incubated with CD8 + T cells purified from OT-I TCR-tg mice specific for OVA 257–264 /H-2K b or OT-II TCR-tg mice specific for OVA 323–339 /H-2IA b at APC:T cell ratios of 1:10, 1:100 and 1:1000. Controls included T cells only with no APC. Control APC included CD11c + DC from spleen. ( a ) Cells for analysis of OT-I responses were collected from cultures and gated as live (PI − ) Thy1.2 + Vα2 + CD8 + T cells. T cell activation was measured at 24 h in terms of % cells expressing CD69. T cell proliferation was measured after 4 days in terms of a reduction in CFSE staining. Data represent mean ± S.E. of three replicate co-cultures. L-DC gave statistically greater T cell activation across all three APC:T cell ratios, while statistically greater T cell proliferation was noted only at an APC:T cell ratio of 10:1 ( p ≤ 0.05). ( b ) Cells for analysis of OT-II responses were collected from cultures and T cells gated as live (PI − ) Thy1.2 + Vα2 + CD4 + cells. T cell activation was measured at 24 h as % cells expressing CD69. T cell proliferation was measured at 4 days in terms of reduction in CFSE staining. Production of regulatory T cells was assessed through intracellular Foxp3 expression in CD4 + T cells analysed after 4 days by flow cytometry

    Article Snippet: Antibodies Fluorochrome-conjugated antibodies specific for CD4 (GK1.5), Thy1.2 (30-H12), CD69 (H12F3), B220 (RA3-6B2), MHC-II (AF6–120.1), F4/80 (C1: A3–1), CD8α (53–6.7), Sirp-α (P84), 4-1BBL (TKS-1), streptavidin-PE-Cy7, streptavidin-PE and streptavidin-FITC were obtained from Biolegend.

    Techniques: Produced, Activated Clotting Time Assay, Mouse Assay, Staining, Expressing, Incubation, Purification, Activation Assay, Flow Cytometry, Cytometry

    Silencing CIITA attenuates α-syn-mediated T cell and monocyte entry. a Representative flow cytometry plots display gating of lymphoid cells (left, representative percent of displayed population is given on plot) followed by CD4 and CD8+ T cells (from the CD45 hi CD11b − population, right, mean raw cell number ± SEM is shown on plot) in the ventral midbrains of mice bilaterally injected with AAV2-SYN + LGFP, AAV2-SYN + LVA, or AAV2-SYN + LVE at 4 weeks post-transduction. b Quantification of absolute numbers of CD4 and CD8 T cells from a . c Representative flow cytometry plots showing gating on LY6C hi monocytes (from the CD45 hi CD11b + population, mean raw cell number ± SEM is shown on plot) in the ventral midbrains of mice bilaterally injected with AAV2-SYN + LGFP, AAV2-SYN + LVA, or AAV2-SYN + LVE at 4 weeks post-transduction. d Quantification of the LY6C hi monocytes from c . For a – d , a total of 24 mice were used—each group consisted of 4 samples with 2 ventral midbrains pooled per sample. Equal numbers of male and female mice were used in these experiments. The mean +/− SEM of samples in each group are plotted. A one-way ANOVA with Tukey’s post hoc test was used for statistical analysis. * p

    Journal: Journal of Neuroinflammation

    Article Title: Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease

    doi: 10.1186/s12974-018-1286-2

    Figure Lengend Snippet: Silencing CIITA attenuates α-syn-mediated T cell and monocyte entry. a Representative flow cytometry plots display gating of lymphoid cells (left, representative percent of displayed population is given on plot) followed by CD4 and CD8+ T cells (from the CD45 hi CD11b − population, right, mean raw cell number ± SEM is shown on plot) in the ventral midbrains of mice bilaterally injected with AAV2-SYN + LGFP, AAV2-SYN + LVA, or AAV2-SYN + LVE at 4 weeks post-transduction. b Quantification of absolute numbers of CD4 and CD8 T cells from a . c Representative flow cytometry plots showing gating on LY6C hi monocytes (from the CD45 hi CD11b + population, mean raw cell number ± SEM is shown on plot) in the ventral midbrains of mice bilaterally injected with AAV2-SYN + LGFP, AAV2-SYN + LVA, or AAV2-SYN + LVE at 4 weeks post-transduction. d Quantification of the LY6C hi monocytes from c . For a – d , a total of 24 mice were used—each group consisted of 4 samples with 2 ventral midbrains pooled per sample. Equal numbers of male and female mice were used in these experiments. The mean +/− SEM of samples in each group are plotted. A one-way ANOVA with Tukey’s post hoc test was used for statistical analysis. * p

    Article Snippet: Isolated cells were blocked with anti-Fcy receptor (clone 2.4G2 BD Biosciences) then incubated with fluorescent-conjugated antibodies against CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, BioLegend), MHCII (M5/114.15.2, BioLegend), Ly6C (clone HK 1.4, BioLegend), CD4 (clone GK1.5, BioLegend), and CD8a (clone 53-6.7, BioLegend).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Injection, Transduction