I am having problems culturing my PBMCs after isolation from human blood samples. I am using RPMI-1640 (plus 10% FBS) supplemented with 25ng/ml human recombinant macrophage colony-stimulating factor. But the cells do no seed on the dish after this. I want to differentiate them into M1 and M2 macrophages.
Any assistance would be appreciated.
Hello
PBMCs are primary culture and cannot remain in culture under normal condition. So in order to maintain PBMC in culture you need to supplement the culture medium RPMI 1640 with mitogen like phytohaemagglutinin (PHA) to stimulate proliferation.
You can add 5-10ug/ml PHA in culture medium. You can also use IL-2. I have never used M-CSF. So, I won't be able to comment on that.
Best.
Dear Ridwan !
Please You look also the protocol:
https://journals.sagepub.com/doi/pdf/10.3727/096368910X557164
Cell Isolation and Culture
PBMCs were either seeded in T75 flasks (regular adherence condition) or cultured in suspension in 30-ml bags. Medium used to culture eEPCs consisted of EBM-2 supplemented with 10% autologous human serum, 1% Pen/Strep, 0.04% hygrodrocortisone, 0.1% heparin, and 0.1% ascorbic acid in presence of IGF-1 (50 ng/ml unless otherwise indicated), EGF (10 ng/ml), FGF (50 ng/ml), VEGF (50 ng/ ml), and fibronectin (10 µg/ml unless otherwise indicated).
The separation can be done in either 15 or 50 ml conical tubes. Aliquot 1 ml of Ficoll-Paque PLUS or Histopaque-1077 for every ml of peripheral blood to be processed. Let Ficoll-Paque PLUS or Histopaque-1077 warm to room temperature as density is temperature-dependent. Mix 1 ml of blood with 1 ml of a balanced salt solution, normally PBS. Overlay the mixed-blood solution on top of the Ficoll-Paque PLUS or Histopaque-1077. The ratio should be 2 ml of the mixed-blood + PBS solution for every 1 ml of Ficoll-Paque PLUS or Histopaque-1077. Centrifuge at 400 x g for 35 minutes at room temperature. During this step, the granulocytes, platelets, and RBCs pellet to the bottom of the tube, and the PBMC float over the Ficoll-Paque PLUS or Histopaque-1077. Carefully aspirate the PBMC from the Ficoll-plasma interface.6. Wash the PBMC with PBS at 300 x g at 4 degrees C twice.
Thank you all Sudipta Chakrabarti Alexandr Chernov Malcolm Nobre . Do PBMC proliferate in culture ( I mean the cells multiply in culture?)
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