hES Cell Fixation and Staining

After harvesting the hES cells using a 0.05% trypsin-EDTA solution (PAA Laboratories, Linz, Austria) and washing them with PBS, single hES cell suspensions were fixed using a 1.6% paraformaldehyde (PFA, Sigma-Aldrich) solution for 10 minutes at room temperature (RT) as we described previously [21 (link)]. The cells were washed and stained using a permeabilisation buffer (Foxp3 Staining Buffer Set, e-Biosciences), blocked using a 2% goat serum (PAA Laboratories) in a permeabilisation buffer (15 minutes) and stained with the appropriate antibodies or their isotype control antibodies for 30 minutes at RT. For cell cycle analysis, the cells were stained with DAPI (Sigma-Aldrich Chemicals). Flow cytometry data were acquired with FACSAria using FACSDiva software (BD Biosciences). Cell permeabilisation, fixation and staining, and data acquisition were done on the same day. The populations that were positive or negative for specific markers were selected using density plots according to the population's borders or using specific biological samples (pluripotent or differentiated hES cells) or specific isotype controls.