Quantifying GM-CSF Bioactivity in Macrophages

GM-CSF bioactivity in BAL fluid and GM-CSF receptor function in transduced or WT macrophages was evaluated by measuring GM-CSF-stimulated phosphorylation of STAT5 in BMDMs or LSK cell-derived macrophages using anti-phospho STAT5 antibody (47/Stat5(pY694)) (BD Biosciences) by flow cytometry as previously reported^{1 (link)}. The STAT5 phosphorylation index (STAT5-PI) was calculated as the mean fluorescence intensity of phosphorylated STAT5 staining in GM-CSF-stimulated cells minus that of non-stimulated cells, divided by that of non-stimulated cells, and multiplied by 100. In experiments to quantify GM-CSF bioactivity, WT BMDMs were incubated in BAL fluid containing anti-GM-CSF (22E9, eBioscience) or isotype control antibody (10 μg/ml) for 30 minutes and then evaluated.