Currently our lab performs post thaw viability via trypan blue. We would like to begin using flow cytometry 7-aad for our post thaw viability. Our attempts so far at thawing our samples have led to the samples clumping and not being able to be performed by flow. Does anyone have a procedure they would be willing to share on how you prep your sample for flow to run?
Add a DNase to your media when you thaw the cells to prevent "clumping". Additionally, you can also pass your cells through a 70um cell strainer to remove any remaining cell aggregates prior to acquiring events on the flow cytometer.
If you dilute sample to 1/5 with PBS as soon thawed they will not clump inmediatly. If you have high concentration of cells may be you can dilute it even more, for 7-AAD viability 20.000 events is going to be more than enough so you don’t need many cells. If you let cells for a few hours clumping will occur.
This is the protocol I Usually use:
500 µL of a solution containing 1/250 of 7-AAD (A1310, Life Technologies) and 1/75 of CD45 FITC (555482, BD) was added to 100 µL of the diluted sample and were incubated in ice for 30 minutes at dark
Hope it helps!
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