Hi,
I am trying to find a solution for my problem during the isolation of PBMCs from buffy coat. I am currently using ficoll gradient to isolate PBMCs.
My steps are :
- dilution of buffy coat at 1/2 in PBS (without Ca and Mg)
- put 13mL of ficoll in falcon and add slowly 17mL of diluted buffy coat
- centrifugation during 30 minutes at 400g (1500 rmp) with no brake
- get the ring of leucocytes and proceed 2 washes in PBS (without Mg and Ca) (centrifugation at 200g and 160g)
- proceed to count cells
At this step I have a lot of very small components, probably platelets with my PBMCs.
Then, I isolate monocytes with negative selection (using Monocyte Isolation Kit II). After isolation, I put monocytes in culture but I still have lot of very small components...
Has anyone ever had this problem? How do you prevent it?
Thank you.
Did you lyse contaminating RBC at any step? - also serves as an extra wash step as per the prior comment - in general you'd be much better off getting meaningful advice(s) if you let people know the ultimate purpose of collecting PBMC.
Thank you for your answers,
Vladimir, no, I don't lyse RBC during my isolation .. but I think that is not a problem because after PBMC isolation, (and despite my lot of little unknown components, maybe platelets) I do negative selection of monocytes (with Monocyte Isolation Kit II) and during this step I get white cell pellet (RBC stay with non-monocytes cells fraction) but when I put isolated monocytes in culture I still have these little components...
Do you remove the plasma after the 30min centrifugation step and before you collect the PBMC layer? We always remove as much plasma as possible and then collect the interphase, that way lots of platelets are already removed. We do the washing steps (for removing the rest of platelets) with 50ml PBS, twice at 200g for 10min with brake. I'm not sure what your purpose is, but the platelets will die very fast in culture anyway, so I would not be that worried about contamination.
Thank you Dafna, I will try to remove the plasma after my first centrifugation and I will increase wash volume.
I hope my problem will be solved :)
Julie:
If your first step is centrifuging whole blood with an anticoagulant to obtain buffy coat, I will suggest that you try a first centrifuging cycle at 4 °C and low speed, say 100g or less for 20 minutes. Then, as Dafna mentioned remove all your plasma, which should contain most of the platelets. This a procedure to obtein what we once called a "platelet rich plasma" Good luck.
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