Mouse IFN‑ gamma Antibody

Detection of IFN‑ gamma in Mouse Spleen.

IFN‑ gamma was detected in immersion fixed paraffin-embedded sections of Mouse Spleen using Rat Anti-Mouse IFN‑ gamma Monoclonal Antibody (Catalog # MAB485) at 10 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Rat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC005). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse IFN-gamma by Flow Cytometry

EAE induction in KO mice.(a) Means±s.e.m. of EAE clinical scores of KO and WT mice. *P<0.05 (two-tailed Student's t-test). (b) EAE incidence in KO and WT mice. *P<0.05 (chi-square test). (c) Means±s.e.m. of body weight of KO and WT mice during EAE induction. Body weight of mice on day 10 post-immunization was considered as 100%. *P<0.05 (two-tailed Student's t-test). (d) Means±s.e.m. of cellularity in draining LN and of cells infiltrating the CNS of mice 14 days after MOG immunization. Mouse numbers (n) and P values (paired two-tailed Student's t-test) are indicated. (e,f) Cytokine-producing cells among CD4 cells from draining LN (e) and CNS (f) on days 13–18 after MOG immunization. Left panels: representative dot plots; right panel: bar graphs (means±s.e.m.) summarizing all the results, with mouse numbers and P values (two-tailed Student's t-test) indicated. (g) HE (left column) or Luxol Fast Blue (right column) staining of spinal cords 30 days after MOG immunization. Asterisks indicate cell infiltration. Arrows point to demyelination. (h) Means±s.e.m. of mononuclear cell infiltration scores, demyelination scores and total pathological scores, which is the sum of the first two scores. Mouse numbers (n) and P values (two-tailed Student's t-test) are indicated. (i) Treg cells in naive KO mice on day 17 during EAE induction. Left panel: representative dot plots; right panel: means±s.e.m. of data from three experiments. NS: not significant (two-tailed Student's t-test). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28169274), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IFN-gamma by Flow Cytometry

EAE induction in KO mice.(a) Means±s.e.m. of EAE clinical scores of KO and WT mice. *P<0.05 (two-tailed Student's t-test). (b) EAE incidence in KO and WT mice. *P<0.05 (chi-square test). (c) Means±s.e.m. of body weight of KO and WT mice during EAE induction. Body weight of mice on day 10 post-immunization was considered as 100%. *P<0.05 (two-tailed Student's t-test). (d) Means±s.e.m. of cellularity in draining LN and of cells infiltrating the CNS of mice 14 days after MOG immunization. Mouse numbers (n) and P values (paired two-tailed Student's t-test) are indicated. (e,f) Cytokine-producing cells among CD4 cells from draining LN (e) and CNS (f) on days 13–18 after MOG immunization. Left panels: representative dot plots; right panel: bar graphs (means±s.e.m.) summarizing all the results, with mouse numbers and P values (two-tailed Student's t-test) indicated. (g) HE (left column) or Luxol Fast Blue (right column) staining of spinal cords 30 days after MOG immunization. Asterisks indicate cell infiltration. Arrows point to demyelination. (h) Means±s.e.m. of mononuclear cell infiltration scores, demyelination scores and total pathological scores, which is the sum of the first two scores. Mouse numbers (n) and P values (two-tailed Student's t-test) are indicated. (i) Treg cells in naive KO mice on day 17 during EAE induction. Left panel: representative dot plots; right panel: means±s.e.m. of data from three experiments. NS: not significant (two-tailed Student's t-test). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28169274), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IFN-gamma by Flow Cytometry

Proliferation and differentiation of naive KO CD4 cells into Th1 and Th17 cells.(a) Proliferation of WT and KO naive spleen CD4 cells under Th1 and Th17 conditions was assessed based on CFSE content according to flow cytometry. Experiments were conducted three times, and representative histograms are shown. Grey peaks represent the CFSE content of CD4 cells at day 0. (b) These cells' differentiation into Th1 and Th17 cells was also determined by flow cytometry according to intracellular IFN-gamma and IL-17 positivity (gated on total CD4+). Representative dot plots are shown in the left panel. Means±s.e.m. of data from three experiments are presented as bar graphs in the right panel. Mouse numbers (n) per group are indicated. P values are reported in the bar graphs (two-tailed Student's t-test). (c,d) T-bet and ROR gamma t expression in CD4 cells cultured under Th1 and Th17 conditions or in IFN gamma + or IL-17+ cells was determined by flow cytometry. Experiments were conducted three times. Representative histograms are shown. (e) Th1 and Th17 differentiation of naive spleen CD4 cells (CD45.2 single-positive) derived from WT and KO donors in chimeric mice was analysed by flow cytometry based on their intracellular IFN-gamma and IL-17 expression. Representative dot plots are shown in the left panel. Means±s.e.m. of data from three experiments are presented as bar graphs in the right panel. Mouse numbers (n) per group are indicated. p values are reported in the bar graphs (two-tailed Student's t-test). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28169274), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IFN-gamma by Flow Cytometry

Proliferation and differentiation of naive KO CD4 cells into Th1 and Th17 cells.(a) Proliferation of WT and KO naive spleen CD4 cells under Th1 and Th17 conditions was assessed based on CFSE content according to flow cytometry. Experiments were conducted three times, and representative histograms are shown. Grey peaks represent the CFSE content of CD4 cells at day 0. (b) These cells' differentiation into Th1 and Th17 cells was also determined by flow cytometry according to intracellular IFN-gamma and IL-17 positivity (gated on total CD4+). Representative dot plots are shown in the left panel. Means±s.e.m. of data from three experiments are presented as bar graphs in the right panel. Mouse numbers (n) per group are indicated. P values are reported in the bar graphs (two-tailed Student's t-test). (c,d) T-bet and ROR gamma t expression in CD4 cells cultured under Th1 and Th17 conditions or in IFN gamma + or IL-17+ cells was determined by flow cytometry. Experiments were conducted three times. Representative histograms are shown. (e) Th1 and Th17 differentiation of naive spleen CD4 cells (CD45.2 single-positive) derived from WT and KO donors in chimeric mice was analysed by flow cytometry based on their intracellular IFN-gamma and IL-17 expression. Representative dot plots are shown in the left panel. Means±s.e.m. of data from three experiments are presented as bar graphs in the right panel. Mouse numbers (n) per group are indicated. p values are reported in the bar graphs (two-tailed Student's t-test). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28169274), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IFN-gamma by Flow Cytometry

CpG-2722 treatment increases PS exposure on cancer through induction of cytokines from immune. (A) Mouse splenocytes were stimulated with/without 5 μg/ml of CpG-2722 for 48 h, & collected the condition medium (C.M.). NHRI-HN1 were treatment with/without 5 μg/ml of CpG-2722 or with/without medium containing 25% C.M. for 24 h as illustrated in the top panel. Phosphatidylserine (PS) exposure on NHRI-HN1 were stained by annexin V conjugated APC antibody, & acquired on a FACS Canto II & analyzed using FlowJo software. Bottom left panel: a representative set of histograms. Bottom right panel: Data represent the mean ± SEM (n = 3). (B) Cytokine levels of TNF-alpha, IFN-gamma, & Trail in tumors from the experiment in Figure 5 were measured by ELISA. Data represent the mean ± SEM (n = 5). (C) NHRI-HN1 were treated with 100 ng/ml of TNF-alpha, IFN-gamma, or Trail for 24 h. Levels of PS exposure were stained by annexin V conjugated APC antibody & acquired on a FACS Canto II. Left panel: a representative set of histograms. Right panel: Data represent the mean ± SEM (n = 3). (D) Mouse splenocytes were stimulated with/without 5 μg/ml of CpG-2722 for 48 h, & the C.M. was collected. NHRI-HN1 were incubated with medium containing 25% of the C.M. in the presence of 1 μg/ml of neutralizing antibody to TNF-alpha, IFN-gamma, Trail or their combination as indicated for 24 h. Phosphatidylserine (PS) exposure on NHRI-HN1 were stained by annexin V conjugated APC antibody, & acquired on a FACS Canto II & analyzed using FlowJo software. Bottom left panel: a representative set of histograms. Bottom right panel: Data represent the mean ± SEM (n = 3). *, **, & *** represent statistically significant differences p < 0.05, p < 0.01, & p < 0.001, respectively, compared with the control or as indicated. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37324949), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IFN-gamma by Flow Cytometry

CpG-2722 treatment increases PS exposure on cancer through induction of cytokines from immune. (A) Mouse splenocytes were stimulated with/without 5 μg/ml of CpG-2722 for 48 h, & collected the condition medium (C.M.). NHRI-HN1 were treatment with/without 5 μg/ml of CpG-2722 or with/without medium containing 25% C.M. for 24 h as illustrated in the top panel. Phosphatidylserine (PS) exposure on NHRI-HN1 were stained by annexin V conjugated APC antibody, & acquired on a FACS Canto II & analyzed using FlowJo software. Bottom left panel: a representative set of histograms. Bottom right panel: Data represent the mean ± SEM (n = 3). (B) Cytokine levels of TNF-alpha, IFN-gamma, & Trail in tumors from the experiment in Figure 5 were measured by ELISA. Data represent the mean ± SEM (n = 5). (C) NHRI-HN1 were treated with 100 ng/ml of TNF-alpha, IFN-gamma, or Trail for 24 h. Levels of PS exposure were stained by annexin V conjugated APC antibody & acquired on a FACS Canto II. Left panel: a representative set of histograms. Right panel: Data represent the mean ± SEM (n = 3). (D) Mouse splenocytes were stimulated with/without 5 μg/ml of CpG-2722 for 48 h, & the C.M. was collected. NHRI-HN1 were incubated with medium containing 25% of the C.M. in the presence of 1 μg/ml of neutralizing antibody to TNF-alpha, IFN-gamma, Trail or their combination as indicated for 24 h. Phosphatidylserine (PS) exposure on NHRI-HN1 were stained by annexin V conjugated APC antibody, & acquired on a FACS Canto II & analyzed using FlowJo software. Bottom left panel: a representative set of histograms. Bottom right panel: Data represent the mean ± SEM (n = 3). *, **, & *** represent statistically significant differences p < 0.05, p < 0.01, & p < 0.001, respectively, compared with the control or as indicated. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37324949), licensed under a CC-BY license. Not internally tested by R&D Systems.