Hi, everyone.
I want to isolate mononuclear cells from bone marrow using gradient centrifugation with Ficoll-Paque Premium 1.077g. However, GE's protocol suggests almost 1:1 of ficoll and bone marrow sample, but I believe this proportion may be higher. someone has already increased to 1:2 or 1:3?
I isolate human bone marrow mononucs frequently. The marrow needs to be adequately diluted (I suggest 3 times in PBS), the cells need to be released from the bone fragments (I do this by agitating the diluted sample with a Pasteur pipette or P1000, then the sample needs to be filtered for fragments (we do this by aspirating into a syringe and then injecting very slowly over the ficoll through a 25G syringe. You can dilute in pbs before these steps and centrifuge for 5 min to aspirate the fat at the top of the supernatant. May be different if mouse/rat work.
Hi, we dilute the aspirates (collected w/ heparin) in Hanks w/o Ca,Mg (has some glucose in it) 1:1. Then incubate for 10 at 37C with gentle shaking and this filtered through BD Falcon Cell Strainer. Add same volume ( as original aspirate) of regular Hanks. You could spin gently ( as per Dr. Bryant above) to try and collect fat cell floaters but we don't regularly do this. This suspension is ready for Buoyant density centrifugation....in 15 ml tubes...with 5-6 mls ficoll and 8-9 mls suspension layered above.
Hi, we always make a dilution of the bone marrow with PBS or Hanks medium in a proportion 1:1. Then, in a 15 ml tube, we put 4 ml of Ficoll at the bottom and 8 ml of the mixture (sample+PBS or Hanks) above. For very low sample quantity (i.e. 1 ml of bone marrow) we make a dilution of 1:2 or even 1:3 to leave enough space for mononuclear cells in the tube for easier recollection. We have used this higher proportions many times and it also works perfectly.
Hope it helps!
Dear Bruna,
First, a disclaimer that I do work for STEMCELL Technologies. The recommendations that you have received of the 1:1 dilution are very much in line with what we recommend as well. I would add that including some FBS in the dilution media tends to be beneficial for cell viability. It is important that the bone marrow be fresh to avoid any clumping which might result in sub-optimal results and loss of PBMCs.
We also have some tubes, called SepMate, that allow for standardized and faster preparations. If you are having any issues with your BM preparations, I would be happy to take this conversation offline.
Best of luck!
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