Fig. 8.

IPE cell dox dependence assay and optimized differentiation results a. (TOP) Fold growth, and (BOTTOM) cell viability of IPE cells cultured with various dox concentrations on TCP. For each time point triplicate 200 μl cultures of IPE cells were plated at 5 × 10⁵ cells/ml in a single well of a 96-well plate. Cells were cultured under static conditions at 37 °C and 5% CO₂ without passage, media change, or agitation. For each measurement, cells were stained with 7AAD and mixed with cell counting beads for analysis with flow cytometry. b: IPE cells after two different differentiation methods. Method 1 (TOP): 4 days of co-culture on MS-5 with 0 ng/ml dox. Method 2 (BOTTOM): 2 days of culture on TCP with 125 ng/ml dox, and then 4 days of co-culture on MS-5 with 0 ng/ml dox. For each condition the final cells were analyzed using flow cytometry measuring forward (FSC) and side (SSC) scatter, 7AAD, CD71 (transferrin receptor), and DRAQ5 (nuclear stain)