I'm having a difficult time with harvesting mouse splenocytes. The protocol I'm using is as follows:
The problem is that when I centrifuge again after ACK lysis (Step 7), ALL of my cells form this white clump. I say this because there are no other cells in the solution when I remove the white clump, and I can't break apart the clump by pipetting or vortexing. From what I've read, this could be due to cells dying and releasing DNA, but I still don't know why there would be so much cell death when I add the ACK buffer which should only lyse RBCs at 4 minutes. Has anyone else run into this problem? If so, then I could really use some help here on how to stop forming this white clump.
In case anyone sees this, I just reduced the time to 30sec-1min, and that was enough time to lyse the RBCs without killing the other cell types from the spleen lysate.
This is because of excessive ACK lysis activity on your cells. Try this: Add 2ml ACK/spleen for just 1 min. Then stop the reaction with complete media (media + FBS) instead of PBS. If you still see some red cells after centrifugation, adjust the time of ACK (increasing 30 sec more) accordingly.