Hi I am currently trying to measure mitochondrial function in human peripheral monocytes/PBMCs. Does anyone know the best way to freeze and thaw the blood cells (and culture) before performing experiments. I have learned time is of the essence, strict SOP and RPMI media 1-2 days after thawing helps.
Write to the following email and you may get a good answer:
E-mail: ude.ub@lokinb
Putting the PBMCs in RPMI medium for 16-24h after thawing has had really good results.
Thank you
Hi Julia --
We use the following un-thaw protocol for human PBMCs:
1. Remove sample for thawing from liquid nitrogen and transfer to water bath at 37C
2. Allow for slow defrosting
3. Re-suspend sample in equal volume of Dextran/Albumin at RT (in most cases this should be adding an additional 0.5ml of D/A to achieve a final volume of 1.0ml. Gently mix cells by pipetting.
4. Take out a 20ul aliquot from the cell suspension to count the cells
5. Transfer the cell suspension to a 15ml conical. Bring total volume to 5ml with Dextran/Albumin. Centrifuge at 330 RCF for 10 minutes at RT. Keep the brake on.
1. Carefully remove the tube and remove/discard the supernatant. Loosen the pellet by tapping the tube with your finger. Based on the cell count, re-suspend the cells in supplemented RPMI (5% heat inactivated FBS, 12.5ml of 1M HEPES, 5ml of 100X Pen/Strep) to desired concentration.
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