During a mononuclear cell isolation from bone marrow from a virally infected but successfully treated patient, there was a trail of whitish cells wider at the MNC layer and narrowing down through the Ficoll to the granulocyte/RBC pellet. Has anyone seen this before, and what can we do to avoid it?
I separated PBMC from blood, did not separate them from bone marrow. When we did this, the critical step is to add Ficoll liquid on the top of the whole blood, very softly, to avoid Ficoll down to the whole blood.
It might help dividing your cells on more gradients. We see the same when isolating cells from patients with myeloproliferative disorders.
Hi Valery, That is really an interesting observation to share with, I didn't see this before. I do not know the protocol that you are using, can I ask you if you are diluting the bone marrow, n if so how many folds. Further dilution might help reduce the 'tornado'. Other factor is the speed, slower the better.
Sorry, not much help; look forward for a constructive discussion from other members.
We dilute the marrow with ~2 volumes PBS w/o Ca2+Mg2+ supplemented with 10% FBS and 2mM EDTA. All reagents are at room temp. We use gradual acceleration and deceleration and spin at 20C. We've only seen this once, and all our samples are from effectively treated, virally infected patients.
Thank you all for your responses.
when receive our BM we do 2 washes with DMEM +10%FBS and Lglut
We then overlay the BM phase very gently on top of the ficol layer avoiding disturbing the interphase. We spin for 45min at 445xg. this is followed by careful removal of the monocyte layer followed by 2 washes in medium prior culture. However, occasionaly if we have a "tail" we just repeat the ficol gradient and that seems to solve the problem.
you seem to do the usual process and since it is a rare observation i would just repeat the ficol gradient for the monocyte layer.
I hope that helps
Thank you, Penelope. You seem to have samples and protocols that are the most similar to ours. Hopefully we won't need to use your suggestion, but it looks like an easy fix.
Val
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