Hi,
I need to isolate PBMCs from the buffy coat (2 ml). I first diluted my 2 ml of buffy coat with 4 ml of PBS (without Ca/Mg). Then I poured it over 3mL of Ficoll (being very careful not to mix).
And after 30min 1200g 20ºC I still have my Ficoll at the bottom and my blood at the top. Is this normal??
Thanks for your help
Hello Roxane Bories,
Separation procedures are optimal when both the buffy coat and Ficoll are at 18–20°C, with an acceptable temperature range of 18–26°C.
The temperature at which density gradient separations are carried out are naturally affected by room temperature, centrifuge temperature, temperature of the density gradient media, and temperature of the liquid sample. There can, at times, also be confusion regarding what is meant by room temperature (for instance, in Europe it can be 20ºC and in North America > 20ºC). Densities of Ficoll products decrease with increase in temperature. For example, Ficoll-Paque premium (1.077 g/ml) exhibits a density of 1.0772, 1.0767, and 1.0758 at 20ºC, 22ºC, and 25ºC, respectively.
Therefore, warm the buffy coat (BC) to room temperature if stored at 4⁰C. It is recommended to warm BC to room temperature for 30-60 minutes before processing through Ficoll density gradient medium. Also warm the Ficoll density gradient media to 18°C to 20°C before use.
In addition, centrifuge at 2200 RCF, 20 minutes, 20⁰C with break OFF.
Hope this helps!
Best,
At least in my former lab the #1 mistake was, that somebody in between two isolations was using the centrifuge at a rpm setting and did not set it back to x g/rcf.
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