Anti il10 percp cy5

To exclude dead cells, the cultured Lin cells were stained with a viable fluorescent reactive dye (Invitrogen, Waltham, MA, USA). Additionally, the cells were stained with anti-human hematopoietic lineage antibody cocktail (eBioscience, San Diego, CA, USA), anti-CD45- allophycocyanin (APC, BioLegend), anti-CD294 (CRTH2)-phycoerythrin (PE, Invitrogen), anti-CD127-PE-cy7 (Biolegend), and anti-CTLA4-APC-Cy7 (Invitrogen). For intracellular cytokine staining, Lin cells were stimulated with 50 ng/mL of PMA (Sigma-Aldrich), 1 μg/mL of ionomycin (Sigma-Aldrich), and 1 μg/mL of brefeldin A (Invitrogen) for 4 h. Thereafter, cells were stained with antibodies against surface markers, fixed, permeabilized using intracellular (IC) Fixation Buffer (eBioscience), and stained with anti-IL-5-APC (BioLegend), anti-IL-13-PerCP/Cy 5.5 (BioLegend), and anti-IL-10-PerCP/Cy 5.5 (BioLegend). The cells stained were examined with a FACSCanto II instrument (Becton Dickinson, Los Angeles, CA, USA). For data analysis, BD FlowJo software (BD Bioscience, version 10.9.0) was utilized.