Hi Patricio orrego
I am currently following this method to isolate the monocyte from pbmc . i get wonderful monocyte without platelets and I add M- CSF to convert the monocyte into macrophages
After centrifugation four distinct layers were obtained by ficoll hypaque: top most layer consisting of plasma, middle ring consisting of PBMCs, a layer of Histopaque and the bottom layer consisting of RBCs. The white colored ring (rich in PBMCs) and you can aspirated middle ring of pbmc in a fresh 15 mL centrifuge tube containing DPBS, 3 times the volume of PBMCs. wash the Pellet of pbmc and do centrifugation at 1000x g for 15 minutes at 4-8°C and again re-suspended the celll pellet in 3 ml DPBS and centrifuge at 600x g for 15 min at 4-8°C
suspend the cell pellet in RPMI 1640 ( without FBS ) and it was centrifuged again at 400x g for 15 minutes at 4-8°C. when you do low centrifugation all the platelets will be removed this is a imp step . u can get pure monocyte .finally re-suspend the cellls in your medium .
All the best .
Hi Patricio orrego
What protocol are you following to get monocyte from pbmc after the ficoll step
Hi Kavita, thanks for answering, I used the protocol of Davies and Gordon 2005 (http://www.ncbi.nlm.nih.gov/pubmed/15361658), particularly the routine isolation method (Ficoll-Paque PLUS + adhesion to tissue culture plastic), but when we add MoAM (containing 7.5% HI autologous human fibrin-depleted plasma + RPMI) we see platelet contamination.
Now we think that protocol proposed by Schilberger et al., 2013 (attached file) could be better for this problem, they do not use autologous plasma, but still we did not try.
What do you think about this?.
Thanks in advance.
A simple method is to wash your suspension after harvesting 3 time by culture media or PBS+BSA, the important thing is to centrifuge at 400 g for 5 to 7 minutes at Temperature between 18 - 25 C. You will still have some platelets but not too much.
Good luck
Hi Patricio orrego
I am currently following this method to isolate the monocyte from pbmc . i get wonderful monocyte without platelets and I add M- CSF to convert the monocyte into macrophages
After centrifugation four distinct layers were obtained by ficoll hypaque: top most layer consisting of plasma, middle ring consisting of PBMCs, a layer of Histopaque and the bottom layer consisting of RBCs. The white colored ring (rich in PBMCs) and you can aspirated middle ring of pbmc in a fresh 15 mL centrifuge tube containing DPBS, 3 times the volume of PBMCs. wash the Pellet of pbmc and do centrifugation at 1000x g for 15 minutes at 4-8°C and again re-suspended the celll pellet in 3 ml DPBS and centrifuge at 600x g for 15 min at 4-8°C
suspend the cell pellet in RPMI 1640 ( without FBS ) and it was centrifuged again at 400x g for 15 minutes at 4-8°C. when you do low centrifugation all the platelets will be removed this is a imp step . u can get pure monocyte .finally re-suspend the cellls in your medium .
All the best .
I am agree with Hafid Soualhine.. low centrifugation at 400g will remove most of the platelets
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