Cells were shipped on ice from a collaborator in NY to California, then were immediately stored in liquid N2 until the day of thawing. Cells were diluted in MEF media (DMEM, FBS, penicillin strep), spun down at 300xg for 5 mins and resuspended in hESC media and plated on MEFs. I am preparing to thaw another vial, any tips?
It is best to ask your collaborator for exact protocol.
However, generally, there are some tips. For example,
1) do not add pen-strep because it weakens the cells
2) use hESC media to dilute thawed cells and add Y27632 (important)
3) add attachment proteins (such as iMatrix) and Y27632 to increase viability of cells
4) increase plating density (e.g. if you're using 6cm dish, switch to 3cm)
Yes I did reach out to them and they advised on not using antibiotics or ROCKI. Was wondering if this would be adequate. Thank you for your feedback.
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