Human IL-2 Antibody

Detection of IL‑2 in PBMC's treated with PMA and Ca2+ ionomycin vs untreated PBMC's by Flow Cytometry PBMC's treated with PMA (50ng/mL) and Ca2+ ionomycin (200ng/mL) overnight (A) vs untreated PBMC's (B) were stained with Mouse Anti-Human IL‑2 Monoclonal Antibody (Catalog # mab202) and Mouse Anti-Human CD3 epsilon APC‑conjugated Monoclonal Antibody (Catalog # FAB100A) followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.

Cell Proliferation Induced by IL‑2 and Neutralization by Human IL‑2 Antibody. Human IL 2 Antibody (Catalog # MAB202) neutralizes IL-2-induced proliferation in the CTLL-2 mouse cytotoxic T cell line. The Neuralization Dose (ND₅₀) is typically 5.00-60.0 ng/mL.

IL‑2 in Human PBMCs. IL-2 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) treated with calcium ionomycin and PMA using Mouse Anti-Human IL-2 Monoclonal Antibody (Catalog # MAB202) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Human IL-2 by Flow Cytometry SLE CD4+ T cells are poised to activate IL-2 signaling during Treg differentiation. (A) Naïve CD4+ T cells were isolated from a systemic lupus erythematosus (SLE) patient & matched healthy control (HC) subject, & cultured for 3 days in the presence of anti-CD3/CD28 & TGF-beta (5 ng/ml) with IL-2 (50 IU/ml) or anti-IL-2 (100 or 1,000 ng/ml). The frequency of CD4+CD25+FOXP3+ cells was determined by flow cytometry. Numbers below the plots represent the frequency of CD4+CD25+FOXP3+ Tregs. The dot plots on the left end represent isotype control staining. (B) CD4+ T cells isolated from matched SLE & HC subjects were cultured for 3 days in the presence of anti-CD3/CD28 & TGF-beta (20 ng/ml) with or without IL-2 (100 IU/ml) or anti-IL-2 (100 ng/ml). Total STAT5 & its phosphorylation at tyrosine 694 were detected by immunoblotting. Representative immunoblot staining (left panel). The signal intensity of phospho-STAT5 & total STAT5 was normalized to that of actin. The normalized pSTAT5 signal intensity (middle panel) & the ratio of normalized pSTAT5 signal intensity over normalized STAT5 signal intensity (right panel) from 3 pairs of matched HC & SLE subjects. (C) Untouched T cells from matched SLE & HC subjects were cultured for 3 days without anti-CD3/CD28 stimulation. Expression of CD25 & FOXP3 in CD4+ cells were determined by flow cytometry. Representative flow cytometry dot plots are shown (left panel). Cumulative data of frequency of CD4+CD25+FOXP3+ & CD4+CD25+ cells, mean fluorescence intensity (MFI) of CD25 expression in CD4+ T cells, & the proportion of CD4+CD25+FOXP3+ cells among CD4+CD25+ cells from 17 pairs of matched SLE & HC subjects (right panel). Data were analyzed by a paired two-tailed t-test (*p<0.05, **p<0.01, ****p<0.0001). Image collected & cropped by CiteAb from the following open publication (https://www.frontiersin.org/articles/10.3389/fimmu.2021.635531/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-2 by Western Blot SLE CD4+ T cells are poised to activate IL-2 signaling during Treg differentiation. (A) Naïve CD4+ T cells were isolated from a systemic lupus erythematosus (SLE) patient & matched healthy control (HC) subject, & cultured for 3 days in the presence of anti-CD3/CD28 & TGF-beta (5 ng/ml) with IL-2 (50 IU/ml) or anti-IL-2 (100 or 1,000 ng/ml). The frequency of CD4+CD25+FOXP3+ cells was determined by flow cytometry. Numbers below the plots represent the frequency of CD4+CD25+FOXP3+ Tregs. The dot plots on the left end represent isotype control staining. (B) CD4+ T cells isolated from matched SLE & HC subjects were cultured for 3 days in the presence of anti-CD3/CD28 & TGF-beta (20 ng/ml) with or without IL-2 (100 IU/ml) or anti-IL-2 (100 ng/ml). Total STAT5 & its phosphorylation at tyrosine 694 were detected by immunoblotting. Representative immunoblot staining (left panel). The signal intensity of phospho-STAT5 & total STAT5 was normalized to that of actin. The normalized pSTAT5 signal intensity (middle panel) & the ratio of normalized pSTAT5 signal intensity over normalized STAT5 signal intensity (right panel) from 3 pairs of matched HC & SLE subjects. (C) Untouched T cells from matched SLE & HC subjects were cultured for 3 days without anti-CD3/CD28 stimulation. Expression of CD25 & FOXP3 in CD4+ cells were determined by flow cytometry. Representative flow cytometry dot plots are shown (left panel). Cumulative data of frequency of CD4+CD25+FOXP3+ & CD4+CD25+ cells, mean fluorescence intensity (MFI) of CD25 expression in CD4+ T cells, & the proportion of CD4+CD25+FOXP3+ cells among CD4+CD25+ cells from 17 pairs of matched SLE & HC subjects (right panel). Data were analyzed by a paired two-tailed t-test (*p<0.05, **p<0.01, ****p<0.0001). Image collected & cropped by CiteAb from the following open publication (https://www.frontiersin.org/articles/10.3389/fimmu.2021.635531/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-2 by Flow Cytometry IL-2 expands IL-13+IFN-gamma + CD8+ T cells in systemic lupus erythematosus (SLE). (A) CD4+ and CD8+ T cells from matched SLE and health control (HC) subjects were cultured as described in Figure 2, and IL-13 and IFN-gamma expression was determined by flow cytometry. (B) Cumulative data of mean fluorescence intensity (MFI) of IFN-gamma and the frequency of IFN-gamma + and IL-13+IFN-gamma + cells. Statistical analysis was made by two-way ANOVA followed by Bonferroni’s correction for multiple comparisons (*p<0.05, **p<0.01, ****p<0.0001). Image collected and cropped by CiteAb from the following open publication (https://www.frontiersin.org/articles/10.3389/fimmu.2021.635531/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-2 by Western Blot IL-2 induces STAT6 phosphorylation and GATA3 expression in systemic lupus erythematosus (SLE) CD8+ T cells. (A) CD8+ T cells from matched SLE and health control (HC) subjects were cultured as described in Figure 2. Expression of GATA-3 and phosphorylation of STAT5 at tyrosine 694 and STAT6 at tyrosine 641 were detected by immunoblotting. Representative immunoblot staining was presented. Lo and Hi concentrations of anti-IL-2 denote 100 and 1,000 ng/ml, respectively. (B) The signal intensity of phospho-STAT5, phospho-STAT6, and GATA-3 were normalized to that of actin. Cumulative data from 9 pairs of matched HC and SLE subjects. Data were analyzed by a two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). (C) Pearson’s and Spearman’s correlation analyses were performed to determine the association between the expression of cytokines (IL-13, IL-5, and IFN-gamma ) and transcription factors (phospho-STAT5, phospho-STAT6, and GATA-3). The blue and red plots represent data from HC and SLE patients, respectively. Spearman correlation coefficient was presented for the association between IL-13 and phospho-STAT5, IL-5 and phospho-STAT-5, and IFN-gamma and GATA-3. Pearson correlation coefficient was presented for the remainder of associations. Image collected and cropped by CiteAb from the following open publication (https://www.frontiersin.org/articles/10.3389/fimmu.2021.635531/full), licensed under a CC-BY license. Not internally tested by R&D Systems.