I have to separate PBMC from bovine blood, and I need a lot of cells, so we are collecting 40 ml of whole blood. When I was reading the Ficoll-Plaque PLUS manual I noticed that there is a limit based on the height of the Ficoll (2.4 cm). Have anyone tried to push this limit? How far can I go and still get a good yield of lymphocytes?
As long as you maintain the Ficoll: Blood ratio you can give it a try. We have performed MNC gradient separation from leukemia patients using 50mL Falcon tubes.
I used to use 20mL Ficoll-Paque Plus + 30mL blood. Spin @ 700 x g for 30 minutes, 18C, no acceleration, no brake. These settings enable very good yield of both PBMCs and granulocytes (if you choose to save the pellet).
We have used 15-20 ml Ficoll-Paque PLUS in 50 ml Falcon tubes, with 30-35 ml of PBS-diluted human whole blood or buffy coats (800 g for 30 min no accel/brake). Of note, we'we had some problems in PBMC separation from whole blood in 50 ml falcons, the Ficoll performance seems to be more consistent in 15 ml falcons.
We have routinely used 12.5mL ficol:hypaque 1.077, over layered with 25-35mL diluted blood(50:50; HBSS) for human PBMCs isolation in 50mL falcon tubes, and generally recover 1-2 million PBMC/ml of whole blood from healthy donors.
Thank you all for the answers, I appreciate it. We've run some tests and the best performance for bovine blood in 50 ml conical tubes was 15 mL Ficoll plus 25 mL of diluted blood (10mL of blood + 15mL of PBS).
Luiz Gustavo Nogueira Almeida at what speed did you centrifuge with the Ficoll? Thank you.
Hello Aspen, I believe it's recommended to spin for 20minutes @ 800G. We found that pre-loading the Ficol-hypaque into tubes for separation and keeping them in the fridge, before centrifugation gives a sharper interface - increased density for layering until it reaches RT. The most important step for a clean interface was the initial layering - angle(45-60˚) the 50ml conical tube - touch the surface of the Ficol with the end of the sample pipette, gently draw it back 1-2cm and make a slight dog leg with the Ficol, then gently run the diluted blood down along this trail onto the Ficol straightening the tube as the blood layer increases.
Works beautifully with human blood separation
2400 to 2800 rpm/30 minutes is the best speed for centrifuge with the Ficoll
Hello Aspen, The rpm value is meaningless unless you know the dimensions of the centrifuge rotor?
This is why only the relative centrifugal force g should be quoted in publication, as it is independent of the instruments rotor head size! You need a nonogram to get meaning from a referenced rpm.
Convert g to rpm
If you know the rpm and radius (r), calculate g using:
g = rpm2 x r x 1.118x10-5
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