Human GM-CSF Antibody

GMCSF in Human PBMCs. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) was detected in human peripheral blood mononuclear cells (PBMCs) using Human GM-CSF Monoclonal Antibody (Catalog # MAB215) at 10 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). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Cell Proliferation Induced by GM‑CSF and Neutralization by Human GM‑CSF Antibody. Recombinant Human GM-CSF (Catalog # 215-GM) stimulates proliferation in the TF-1 human erythroleukemic cell line in a dose-dependent manner (orange line) as measured by Resazurin (Catalog # AR002). Proliferation elicited by Recombinant Human GM-CSF (0.1 ng/mL) is neutralized (green line) by increasing concentrations of Human GM-CSF Monoclonal Antibody (Catalog # MAB215). The ND₅₀ is typically 0.075-0.45 µg/mL.

Detection of GM-CSF by Flow Cytometry TC-derived soluble factors promoted neutrophil survival. A. Neutrophils were cultured in a TC-CM or the control medium. At the indicated time points, live cells were evaluated by flow cytometry with FITC-conjugated annexin V and PI. Results were expressed as percentages of live cells (mean ± SEM of five independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05. B. Representative flow cytometric panels of dot plots of PMNs cultured in a TC-CM or control medium and stained with FITC-conjugated annexin V and propidium iodide (PI) at 24 (upper panels) and 48 (lower panels) hours. C. The GM-CSF release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results were expressed as mean ± SEM of seven independent experiments; ****p < 0.001; ***p < 0.005. D-F. Neutrophil survival in a TPC1-derived (D-E) or 8505c-derived (F-G) conditioned medium was evaluated in the presence of an anti-GM-CSF blocking antibody or the relative isotype control (10 μg/ml). At 24 hours, live cells were stained with FITC-conjugated annexin V and PI and analyzed by flow cytometry. Figs E and G illustrate representative flow cytometric panels of one out of five independent experiments. The results were expressed as mean ± SEM of five independent experiments; **p < 0.01; *p < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29953504), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of GM-CSF by Immunohistochemistry Knockdown of GM-CSF protein levels after siRNA application in cancer cells. HeLa/DLD-1 cells were transfected with control siRNA (1/1*, 2/2*) or GM-CSF siRNA (3/3*, 4/4*) and cultured in the absence or presence of 25 ng/mL HB-EGF. The numbers indicate the culture conditions and the corresponding supernatants (SN) used for ELISA or cell stimulation. (A) Blockade of GM-CSF production in cultures of HeLa/DLD-1 cells transfected with GM-CSF siRNA was confirmed by immunocytochemistry (2/2* vs. 4/4*) and ELISA (left side; 2/2* vs. 4/4*, p < 0.05). (B) SN from GM-CSF-silenced HeLa/DLD-1 did not induce HB-EGF expression in mononuclear phagocytes (Mø), as revealed by flow cytometry (2/2* vs. 4/4*) and ELISA (left side; 2/2* vs. 4/4*, p < 0.05). (C) Mø stimulated with SN from GM-CSF-silenced HeLa/DLD-1 cells released SN less effective at inducing GM-CSF in non-silenced cancer cells, as determined by ELISA (see Methods section; SN2 vs. SN4, p < 0.05). Representative pictures or the means ± SD out of 5 experiments are shown. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/20946648), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of GM-CSF by Flow Cytometry TC-derived soluble factors promoted neutrophil survival. A. Neutrophils were cultured in a TC-CM or the control medium. At the indicated time points, live cells were evaluated by flow cytometry with FITC-conjugated annexin V and PI. Results were expressed as percentages of live cells (mean ± SEM of five independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05. B. Representative flow cytometric panels of dot plots of PMNs cultured in a TC-CM or control medium and stained with FITC-conjugated annexin V and propidium iodide (PI) at 24 (upper panels) and 48 (lower panels) hours. C. The GM-CSF release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results were expressed as mean ± SEM of seven independent experiments; ****p < 0.001; ***p < 0.005. D-F. Neutrophil survival in a TPC1-derived (D-E) or 8505c-derived (F-G) conditioned medium was evaluated in the presence of an anti-GM-CSF blocking antibody or the relative isotype control (10 μg/ml). At 24 hours, live cells were stained with FITC-conjugated annexin V and PI and analyzed by flow cytometry. Figs E and G illustrate representative flow cytometric panels of one out of five independent experiments. The results were expressed as mean ± SEM of five independent experiments; **p < 0.01; *p < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29953504), licensed under a CC-BY license. Not internally tested by R&D Systems.