Hi,
I recently cultured the AES0186-BRC-6 Feeder free mESc. When I cultured the cells, they were good until the second passage, and at the third passage, the cells died. I used the following media and cultured the cells on 0.1% gelatin:
Reagents: DMEM(1X) + GLUTAMAX (High Glucose)(Gibco31966-021), 15 % Knockout Serum Replacement (Fisher Scientific# 1614107975), NEAA (0.1mM) (Gibco11140050), 2-b mercaptoethanol 0.1mM (Gibco21985023), LIF (Thermo Fisher# A35933, 1000 U/ml), Pen/Strep (Gibco15140-1635 ml (1x)), GSK-3 inhibitor (EnzoENZ-CHM124, (2uM)). I used Accutase for dissociation. Can you please guide me if there is anything wrong with the media, or what can I do to ensure that the cells remain healthy? The plating density was 25,000 cells/cm2.
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