Macsquant analyser flow cytometer

hPSCs were harvested as single cell suspensions, fixed with 2% para-formaldehyde, then stored in 10% FBS in PBS. For detection of extracellular antigens, hPSCs were kept in 10% FBS in PBS. For detection of intracellular antigens, hPSCs were resuspended in 0.1% saponin permeabilization buffer in 10% FBS in PBS for 15 min [15 (link),51 (link)]. hPSCs were incubated for 20 min with the following primary antibodies: 2 μg/μL anti-TRA-1-60 and 2 μg/μL anti-TRA-1-81 (Abcam, Cambridge, UK); or 0.25 μg/μL anti-OCT4 antibody (BD Biosciences). The anti-TRA antibodies were detected using Alexa Fluor-488 anti-mouse IgM secondary antibody (Thermo Fisher Scientific, Sydney, Australia), and the anti-OCT4 antibody using Alexa Fluor-488 anti-mouse IgG antibody (Thermo Fisher Scientific). Samples were analyzed using a MACSQuant^® Analyser Flow Cytometer (Miltenyi Biotec, North Ryde, Australia). Biological replicates for each sample were averaged, and comparisons were made using the Student’s t-test.

The cell populations in BAL were characterized by their specific surface markers [33 (link)] as described in [24 (link)]. Briefly, BAL fluid was centrifuged at 1,000 g for 10 min. The resulting cells were suspended in PBS (10⁵ in 100 μL PBS), transferred to low-binding polypropylene 96-well plates (Corning; Avon, France) and analysed with a MACSQuant^® Analyser flow cytometer (Miltenyl Biotec); data for at least 10,000 events were recorded.