normal rat igg2b  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Crosstalk between microbiome, regulatory T cells and HCA2 orchestrates the inflammatory response in a murine psoriasis model

doi: 10.3389/fimmu.2023.1038689

Figure Lengend Snippet: HCA2 signaling regulates the suppressive activity in vitro and the phenotype of Treg. (A) CD4 + CD25 + (Treg) cells were isolated from pooled spleens and lymph nodes (LN) of 7 mice and subjected to FACS analysis. The cell population from both strains was analyzed for double positive CD25/Foxp3 cells and is demonstrated as percentage and total number of double positive cells. As negative control served isotype (IgG Co). FACS analysis is also shown as a scatter graph with mean ± SD. Y axes show percentage of double positive cells. Data were analyzed by using the t test with Welch´s correction. n = 3 (B) Pooled LN cells and splenocytes of 4 mice, obtained from both strains were analyzed for double positive Foxp3/IL-10, Foxp3/GARP and Foxp3/CD25 cells and is demonstrated as percentage and total number of double positive cells. FACS analysis is also shown as a scatter graph with mean ± SD. Y axes show percentage of double positive cells. Data were analyzed by using the t test with Welch´s correction. n = 3 Data are presented from one of three independent experiments. (C) Pooled LN and spleen cells obtained from WT and HCA2-KO mice (4 for each group) were separated into CD4 + CD25 - (responder cells) and CD4 + CD25 + cells (Treg). Treg and responder cells were mixed at the ratios 1:1, 1:2 and 1:4. Responder cells were activated and after 4 days, cell proliferation was measured using Cell Counting Kit-8. Data are presented as percent suppression from one of three independent experiments. Student t test and one-way ANOVA was performed. *P < 0.03 WT vs HCA2-KO 4:1; P = 0.11504 WT vs HCA2-KO 2:1; ns, not significant; **P < 0.04 WT vs HCA2-KO 1:1; P ANOVA = 0.016; n = 4.

Article Snippet: As isotype controls the following immunoglobulins: rat IgG2a-APC (Cat #sc-2894, RRID: AB_737246) as control for Foxp3-APC; rat IgG2b-APC (Cat #sc-2895, RRID: AB_737266) as control for IL-10-APC and CD4-APC (both from Santa Cruz); rat IgG2a-PE (Becton Dickinson; Cat #553930, RRID: AB_479719) as control for GARP-PE and Foxp3-PE; rat IgG2b-PE (Cat #556925, RRID: AB_479625) as control for IL-10-PE; rat IgG1-APC (Cat #400411, RRID: AB_326517) as control for IL-6-, IL-17-, IL-23-APC; rat IgG1-PE (Cat #400407, RRID: AB_326513) as control for IL-6-, CD25-, IL-17- and IL-23-PE (both from BioLegend); rat IgG2b-PE (Cat # 556925, RRID: AB_479625) as control for PD-1 and Ki-67-PE (Becton Dickinson); armenian hamster IgG1-PE (Becton Dickinson; Cat #553972, RRID: AB_395172) and Alexa-488 conjugated (BioLegend; Cat #400923; RRID: AB_2814703) as control for CTLA-4-PE, and Helios-A488; rat IgG1-PE (Miltenyi Biotec, Cat # 130-123-746, RRID: AB_2857627) as control for CD73- and FR4-PE.

Techniques: Activity Assay, In Vitro, Isolation, Negative Control, Cell Counting

Journal: Frontiers in Immunology

Article Title: Crosstalk between microbiome, regulatory T cells and HCA2 orchestrates the inflammatory response in a murine psoriasis model

doi: 10.3389/fimmu.2023.1038689

Figure Lengend Snippet: HCA2 deficiency causes defects of Treg at the site of inflammation. (A) Treg obtained from 7 HCA2-KO and 7 WT mice were stained with CFSE and injected i.v. into IMQ-treated WT animals. After 48 h LN and spleens were obtained from the recipient mice and FACS analysis of CFSE-negative (overlay of red histograms) or CFSE-positive cells (overlay of green histograms) was conducted. The expression of IL-6, IL-17, IL-23 and IL-10 was standardized on isotype controls. For green histograms (Treg from WT and HCA2-KO) two isotype controls were used. For IL-6, IL-17 and IL-23 the same IgG (rat IgG1) control was used. Histograms show fluorescence intensity (x axis) versus cell count (y-axis). The number of cells is displayed in the histograms. (B) FACS analysis is also shown as scatter graph with mean ± SD. Y axes show percentage of positive cells. Data were analyzed by using the t test with Welch´s correction. For multiple comparisons we carried out one-way ANOVA in order to assess whether at least two groups significantly differ. 1 WT +IMQ + Treg from WT (red); 2 WT +IMQ + Treg from WT (green); 3 WT +IMQ + Treg from HCA2-KO (red); 4 WT +IMQ + Treg from HCA2-KO (green) n=3.

Article Snippet: As isotype controls the following immunoglobulins: rat IgG2a-APC (Cat #sc-2894, RRID: AB_737246) as control for Foxp3-APC; rat IgG2b-APC (Cat #sc-2895, RRID: AB_737266) as control for IL-10-APC and CD4-APC (both from Santa Cruz); rat IgG2a-PE (Becton Dickinson; Cat #553930, RRID: AB_479719) as control for GARP-PE and Foxp3-PE; rat IgG2b-PE (Cat #556925, RRID: AB_479625) as control for IL-10-PE; rat IgG1-APC (Cat #400411, RRID: AB_326517) as control for IL-6-, IL-17-, IL-23-APC; rat IgG1-PE (Cat #400407, RRID: AB_326513) as control for IL-6-, CD25-, IL-17- and IL-23-PE (both from BioLegend); rat IgG2b-PE (Cat # 556925, RRID: AB_479625) as control for PD-1 and Ki-67-PE (Becton Dickinson); armenian hamster IgG1-PE (Becton Dickinson; Cat #553972, RRID: AB_395172) and Alexa-488 conjugated (BioLegend; Cat #400923; RRID: AB_2814703) as control for CTLA-4-PE, and Helios-A488; rat IgG1-PE (Miltenyi Biotec, Cat # 130-123-746, RRID: AB_2857627) as control for CD73- and FR4-PE.

Techniques: Staining, Injection, Expressing, Control, Fluorescence, Cell Counting

Journal: Frontiers in Immunology

Article Title: Crosstalk between microbiome, regulatory T cells and HCA2 orchestrates the inflammatory response in a murine psoriasis model

doi: 10.3389/fimmu.2023.1038689

Figure Lengend Snippet: HCA2 signaling regulates the suppressive activity in vitro and the phenotype of Treg. (A) CD4 + CD25 + (Treg) cells were isolated from pooled spleens and lymph nodes (LN) of 7 mice and subjected to FACS analysis. The cell population from both strains was analyzed for double positive CD25/Foxp3 cells and is demonstrated as percentage and total number of double positive cells. As negative control served isotype (IgG Co). FACS analysis is also shown as a scatter graph with mean ± SD. Y axes show percentage of double positive cells. Data were analyzed by using the t test with Welch´s correction. n = 3 (B) Pooled LN cells and splenocytes of 4 mice, obtained from both strains were analyzed for double positive Foxp3/IL-10, Foxp3/GARP and Foxp3/CD25 cells and is demonstrated as percentage and total number of double positive cells. FACS analysis is also shown as a scatter graph with mean ± SD. Y axes show percentage of double positive cells. Data were analyzed by using the t test with Welch´s correction. n = 3 Data are presented from one of three independent experiments. (C) Pooled LN and spleen cells obtained from WT and HCA2-KO mice (4 for each group) were separated into CD4 + CD25 - (responder cells) and CD4 + CD25 + cells (Treg). Treg and responder cells were mixed at the ratios 1:1, 1:2 and 1:4. Responder cells were activated and after 4 days, cell proliferation was measured using Cell Counting Kit-8. Data are presented as percent suppression from one of three independent experiments. Student t test and one-way ANOVA was performed. *P < 0.03 WT vs HCA2-KO 4:1; P = 0.11504 WT vs HCA2-KO 2:1; ns, not significant; **P < 0.04 WT vs HCA2-KO 1:1; P ANOVA = 0.016; n = 4.

Article Snippet: As immunoglobulin controls rat IgG2b FITC and rat IgM APC (both from Santa Cruz Biotechnology, Dallas, USA, Cat #sc-2835, RRID: AB_737268; Cat #sc-2896, RRID: AB_737296) were used.

Techniques: Activity Assay, In Vitro, Isolation, Negative Control, Cell Counting

Journal: Frontiers in Immunology

Article Title: Crosstalk between microbiome, regulatory T cells and HCA2 orchestrates the inflammatory response in a murine psoriasis model

doi: 10.3389/fimmu.2023.1038689

Figure Lengend Snippet: HCA2 deficiency causes defects of Treg at the site of inflammation. (A) Treg obtained from 7 HCA2-KO and 7 WT mice were stained with CFSE and injected i.v. into IMQ-treated WT animals. After 48 h LN and spleens were obtained from the recipient mice and FACS analysis of CFSE-negative (overlay of red histograms) or CFSE-positive cells (overlay of green histograms) was conducted. The expression of IL-6, IL-17, IL-23 and IL-10 was standardized on isotype controls. For green histograms (Treg from WT and HCA2-KO) two isotype controls were used. For IL-6, IL-17 and IL-23 the same IgG (rat IgG1) control was used. Histograms show fluorescence intensity (x axis) versus cell count (y-axis). The number of cells is displayed in the histograms. (B) FACS analysis is also shown as scatter graph with mean ± SD. Y axes show percentage of positive cells. Data were analyzed by using the t test with Welch´s correction. For multiple comparisons we carried out one-way ANOVA in order to assess whether at least two groups significantly differ. 1 WT +IMQ + Treg from WT (red); 2 WT +IMQ + Treg from WT (green); 3 WT +IMQ + Treg from HCA2-KO (red); 4 WT +IMQ + Treg from HCA2-KO (green) n=3.

Article Snippet: As immunoglobulin controls rat IgG2b FITC and rat IgM APC (both from Santa Cruz Biotechnology, Dallas, USA, Cat #sc-2835, RRID: AB_737268; Cat #sc-2896, RRID: AB_737296) were used.

Techniques: Staining, Injection, Expressing, Control, Fluorescence, Cell Counting