Measuring ALDH Activity and Cytokine Production in Cells

ALDH activity in individual cells was estimated using ALDEFLUOR staining kits (Stemcell Technologies) as previously described14 (link). Cells were stained with indicated mAbs conjugated to various fluorochromes in the presence of anti-CD16/CD32 mAb. Intracellular staining of Foxp3 was performed using mouse/rat Foxp3 staining sets (eBioscience). For intracellular cytokine staining, CD4⁺ T cells obtained from cultures were restimulated for 5 h with PMA (50 ng/ml) and ionomycin (750 ng/ml) (both from Calbiochem). Monensin (3 μM; Sigma-Aldrich) was added to the cultures for the last 2 h. After surface staining, the cells were fixed with Fixation Buffer (BioLegend), and intracellular cytokine staining was performed according to the manufacturer’s protocol. Analysis was performed on a FACSAria or FACSCalibur with CellQuest Pro software (BD Biosciences).