Sc 21702

Human PSCs were characterized as described previously^{84 (link)}. Briefly, the hPSCs were monitored regularly microscopically, and characterized by immunofluorescence staining (IHC) for expression of pluripotency markers using the following primary antibodies; Nanog (1:200, R&D Systems, AF1997), OCT-3/4 (1:200, R&D Systems, AF1759), SSEA-3 (1:300, Novus Biologicals NB100–1832), SSEA-4 (1:200, R&D Systems, MAB1435), TRA-1-60 (1:200, Millipore, MAB4360), TRA-1-81 (1:200, Santa Cruz Biotechnology SC-21706), and early marker for differentiation SSEA-1 (1:200, Santa Cruz Biotechnology, SC-21702). Alexa Fluor-conjugated (1:400, ThermoFisher Scientific A-11055, A-21042, A-10037), and FITC-conjugated (1:400, Novus Biologicals, NB7102) secondary antibodies were used. Nuclei were counterstained with 4′,6′diamidino-2-phenylidole (DAPI) (Vector Laboratories Inc., Burlingame, CA).
Pluripotency was verified with in vitro pluripotency assay by spontaneous differentiation as embryonic bodies^{84 (link)}, followed by immunofluorescence analysis for alpha-smooth muscle actin (SMA, 1:400, R&D Systems, MAB1420) for mesoderm, alpha-fetoprotein (AFP, 1:200, R&D Systems MAB1369) for endoderm, and OTX2 (1:200, R&D Systems, AF1979) for ectoderm. Karyotyping was performed at Finnish Microarray and Sequencing Centre (FMSC), Turku Centre for Biotechnology with the KaryoLite BoBs assay (Perkin Elmer).

Cells were fixed in 4% paraformaldehyde for 30 min and incubated at 37°C in blocking buffer (PBS containing 5% BSA and 0.2% Triton X-100). Cells were incubated in the presence of primary antibodies at 4°C overnight and then washed three times with PBS. Cells were then incubated with the Alexa Fluor 488 (Invitrogen, 1:1000) secondary antibody for 1 h at 37°C. Nuclei were stained with Hoechst (Invitrogen, 1:5000). The primary antibodies and dilutions used were Oct4 (sc-5279, Santa Cruz Biotechnology, 1:200), SSEA1 (sc-21702, Santa Cruz Biotechnology, 1:100) and Nanog (ab808692, Abcam, 1:100).