Anti ssea3 antibody

The cells were fixed with paraformaldehyde solution in PBS (4% w/v), permeabilized with Triton X-100 (0.1%), treated with bovine serum albumin (Affimetrix, USA) in PBS (3%) for 1 h to block the nonspecific binding, washed in PBS thrice for 15 min, incubated with Anti-SSEA3 antibody (Abcam, UK) and Anti-CD105 antibody (BD, USA) overnight at 4 °C, washed with PBS, exposed to Alexa Fluor 488 goat anti-rat antibody (Invitrogen Life Technologies, USA) for 1 h at 37 °C, and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Images of the stained cells were obtained using an FV1000 Olympus confocal microscope (Olympus, Japan).

Cell surface antigens for human iPSCs were analyzed with FACS. Cells (1 × 10⁵ cells per well) were incubated with one of the following primary antibodies: anti-CD24 antibody (Abcam), 1:100 dilution; anti-SSEA3 antibody (Abcam), 1:100 dilution; anti-CD105 antibody (Abcam), 1:200 dilution; anti-Nanog antibody (CST), 1:100 dilution; anti-Sox2 antibody (CST), 1:300 dilution; and anti-Oct4 antibody, 1:600 dilution (CST). After 1 h of incubation at 4 °C, the cells were washed, centrifuged, stained with 100 μl of a secondary antibody selected from anti-mouse IgG (H + L) Alexa Fluor® 488 Conjugate (CST) at 1:1000 dilution, anti-rat IgG (H + L) Alexa Fluor® 488 Conjugate (CST) at 1:1000 dilution, and anti-rabbit IgG (H + L) Alexa Fluor® 488 Conjugate (CST) at 1:1000 dilution. The stained cell pellets were suspended in 200 μl PBS/10 % FCS at 4 °C for flow cytometry analysis. All antibodies were validated for antigen specificity.