Figure 4.

Flow cytometry analysis of mES cell viability after a 30-min incubation in LCB. Routinely cultured mES cells were trypsinized, centrifuged, resuspended in LCB, and kept for 30 min either on ice or at room temperature (RT). Cells were then transferred to adhesion culture on fibronectin-coated plates supplemented with the ES medium, and maintained in a tissue culture incubator. Cells were sampled immediately after the 30-min LCB incubation and 24 h and 72 h thereafter, and analyzed for viability using 7-AAD staining. An equal mixture of healthy and dead mES cells was used to define a gating for the live population (“Gating Control”). Routinely maintained mES cells without LCB incubation were used as a positive control (“ES Medium”). Based on percentage of viable cells, no adverse effects on cell viability due to 30-min LCB incubation were observed. FSC-A: forward scattering area; PerCP-Cy5-5-A: light intensity corresponding to 7-AAD’s emission