PBMC proliferation assay

The capacity of ASC or their bioproducts to inhibit induced proliferation of peripheral blood mononuclear cells (PBMCs) was analyzed as described before [27]. PBMCs were isolated from leukapheresis samples from healthy donors, provided by the German Red Cross Blood Donor Service in Mannheim (Mannheim Ethics Commission; vote number 2018-594 N-MA). To assess their proliferation, PBMCs were labelled with proliferation dye Cytotell Green (ATT Bioquest, 22,253) (1:500 dilution) and seeded at a 1:10 ASC/bioproduction:PBMCs ratio in RPMI, supplemented with 10% FBS, 2% l-glutamine (PAN Biotech, P04-80100), 1% Penicillin/Streptomycin (PAN Biotech, P06-07100), and 200 U/mL IL-2 (Promokine, {"type":"entrez-nucleotide","attrs":{"text":"C61240","term_id":"2419945","term_text":"C61240"}}C61240). PBMC proliferation was stimulated with phytohemagglutinin-L (PHA, 4.8 µg/mL (Biochrom, Merck Millipore, M5030)). PBMCs cultured alone without ASC in the absence and presence of PHA served as negative and positive controls, respectively. After 5 days, PBMC proliferation was measured based on the dilution of Cytotell Green dye using a FACS Canto II (BD Biosciences) and the data were analyzed with FlowJo Software.