Hi all,
I am struggling to obtain any iPSc colonies with my routine reprogramming by infection. To give you more background my cells are MEFs p53 KO with a floxed allele for TRF1, an ubicuous Cre recombinase, and an inducible dox OSKM cassette (that I do not use as I obtain more efficiency by infeccion).
The problem is the moment I infect (I used brand new pCL-Eco and OSK plasmids), using sequential infection (2 times a day for 2 days from 293T supernatant with packaged virus plus polybrene) my cells start to die forming the structures that I show in the picture.
Has anyone observed this before? Do you have any idea why they could die from just infection?
Pd: The control cells in parallel, look nice (2nd image)
Thank you in advance!!
Hi,
There can be two main reasons for this situation:
1. Too many virus particles (optimize the number of virus particles);
2. Mycoplasma infection (do a mycoplasma test and do a treatment before virus addition).
You can increase cell viability after reprogramming by adding a ROCK inhibitor.
Justyna Augustyniak
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