To confirm that bank cultures are undifferentiated and self-renewing, WiCell will use flow cytometry to assess the presence of both intracellular and extracellular markers indicative of self-renewal post-thaw.  WiCell utilizes a small, two-laser, four-detector flow cytometer (AccuriC6, BD Biosciences, San Jose, CA) that allows the detection of four fluorochromes simultaneously (Alexa488, PE, 7AAD, APC).  WiCell’s standard flow cytometry characterization panel to assess differentiation status includes 7AAD (DNA intercalating dye), Oct4 (intracellular marker of undifferentiated status/self-renewal), and SSEA4, SSEA3, Tra 1-60 and Tra 1-81 (cell surface markers of undifferentiated status/self-renewal) as well as SSEA 1 (cell surface marker in differentiated cells).  The combination of these antibodies/fluorochromes will allow the identification of undifferentiated and/or differentiating cells in the same test.  Additionally, the intensity of each antigen can be measured over time with the use of a flow cytometer that always maintains the same sensitivity.  Without this multi-color and standard approach these comparisons could not be made.  For this assay, a standard report indicating the percentage of cell positive for each individual marker, and the percent of cells staining positive for both Oct4 and SSEA4 simultaneously is provided.  Assessment of the expression of these markers in combination allows a quantitative assessment of the percentage of undifferentiated cells in the starting iPSC population and allows assessment of the population heterogeneity.  Additional markers may be added upon request.

When you assay is complete you will receive a copy of an Assessment of Undifferentiated Status report.  Please see our sample Assessment of Undifferentiated Status report.