Anti ssea3

Immunochemistry was performed as previously described [29 (link)]. The primary antibodies used were anti-Oct4 (1:100, Santa Cruz Biotechnology), anti-Nanog (1:150, Santa Cruz Biotechnology), anti-Tra-1-60 (1:150, Chemicon), anti-SSEA3 (Ascites, 1:400, Developmental Studies Hybridoma Bank), anti-CIITA (1:200, Santa Cruz Biotechnology) and anti-HLA DR+DP+DQ (1:200, Abcam) for staining.

Following the 7 day differentiation period a single cell suspension was obtained through the trypsinisation of monolayers and cells were resuspended in blocking buffer (0.1% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS)) at a density of 0.2 × 10⁶ cells per well of a 96 well plate (Greiner Bio-one, Stonehouse, UK). The plate was centrifuged at 1000 rpm for 3 min at 4 °C, and the pellet was resuspended in the appropriate primary antibody (anti-p3X (gift from Prof. Peter Andrews, Sheffield University, UK), anti-SSEA3 (Developmental Studies Hybridoma Bank, Iowa, USA), anti-TRA-160 (Millipore, Darmstadt, Germany) or anti-A2B5 (R&D Systems, Abingdon, UK)) for 60 min. The cells were then washed 3 times in blocking buffer prior to incubation with the secondary antibody (anti-mouse IgM (Sigma-Aldrich)) for 60 min. Cells were then washed a further three times in blocking buffer and resuspended in blocking buffer prior to analysis using the Guava Easy Cyte Plus and Cytosoft software (Millipore), and settings adjusted to the negative control (P3X), a murine marker that is not be expressed on human cells to take non-specific staining into account.