Human IL-2 Antibody

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 # MAB202R) at 25 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.

Cell Proliferation Induced by IL-2 and Neutralization by Human IL-2 Antibody. Recombinant Human IL-2 (Catalog # 202-IL) stimulates proliferation in the CTLL-2 mouse cytotoxic T cell line in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Human IL-2 (2 ng/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human IL-2 Monoclonal Antibody (Catalog # MAB202). The ND₅₀ is typically 0.015-0.03 µg/mL.

Detection of IL-2 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) (A) treated with Cell Activation Cocktail 500x (5476) for 5 hours or (B) resting were stained with Mouse Anti-Human IL-2 Monoclonal Antibody (Catalog # MAB202R) followed by Goat anti-Mouse IgG APC-conjugated Secondary Antibody (F0101B) and Mouse anti-Human CD3 PE-conjugated Monoclonal Antibody (FAB100P). Quadrant markers were set based on Mouse IgG1 isotype control antibody (MAB002). To facilitate intracellular staining, cells were fixed and permeabilized using FlowX FoxP3/Transcription Factor Fixation & Perm Buffer Kit (FC012). Staining was performed using our Staining Intracellular Molecules protocol.

Detection of Human IL-2 by Flow Cytometry IL-2 induces IL-32 but TCR activation is required for IL-32 secretion. (A) IL-32 expression in CD3+ T from healthy donors (HDs) after 24 h of resting (unstimulated) either alone or in the presence of IFN-gamma & low (100 IU/ml) or high (3,000 IU/ml) amounts of IL-2 & after 24 h of either CD3 or CD3/CD28 stimulation determined by ICFC analysis. Representative data from n=2 independent experiments. (B–D) WB analyses of IL-32 expression in cell lysates & IL-32 secretion into the cell culture supernatants (enriched via Amicon Ultracentrifugal filters) by HD CD3+ T after treatment for 48 h with 500 or 3,000 IU/ml IL-2 alone (low or high IL-2, respectively) or treatment with high IL-2 for 48 h with subsequent CD3/CD28 stimulation for 72 (h) (B) Representative blot of lysates & supernatants (15 µg) from n=2 HDs (#1, #2) next to rIL-32 beta (4 ng, MW: 23.1 kDa) & rIL-32 gamma (4 ng, MW: 28.1 kDa). As a loading control, the housekeeping gene GAPDH (MW: 37 kDa) was detected on same blot. (C) Cumulative data of quantified IL-32 beta expression in cell lysates normalized to GAPDH for n=2–5 HDs, mean+SD, dots depict data from individual experiments, Student’s paired t-test, *p<0.05. (D) Cumulative data of quantified IL-32 beta secretion into culture supernatants displayed by the Area Under the Curve (AUC) for n=2–5 HDs, mean+SD. (E, F) IL-32 expression in HD T after resting (Unstim) or CD3/CD28 stimulation alone (Stim) or together with either an alpha IL-2 neutralizing antibody (Stim + alpha IL-2) or the respective isotype control (Stim + Isotype) for 24 (h) (E) Representative dot plots depicting IL32+CD25+ among live single T stimulated in the presence of the isotype or the alpha IL-2 antibody. (F) Cumulative data showing the frequency of IL-32+CD25+ among live single T, n=2 HDs, Student’s unpaired t-test, *p<0.05. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39211051), licensed under a CC-BY license. Not internally tested by R&D Systems.