My problem is that I have to isolate PBMCs through ficoll manually. and then I use them for positive selection of T lymphocytes by MACS (with anti-CD3 antibody). The only material we have is ficoll. what is the best protocol for me in this condition?
Hello Zahra Bagherpour
Ficoll-Paque will give good results for PBMC isolation. It provides a density gradient medium containing sodium diatrizoate, polysaccharides, and water, and has a density of 1.077 g/mL. This medium is denser than lymphocytes, monocytes, and platelets (meaning these will remain above it), but less dense than granulocytes and erythrocytes, which will drop below it.
The function of sodium diatrizoate in ficoll-paque is to provide the optimal density and osmolarity necessary for the efficient removal of other cells from the mononuclear cells.
If you have Ficoll in powdered form in the laboratory, you will need to add sodium diatrizoate so as to increase Ficoll's density and hence better separation.
You could try the following composition for Ficoll-Paque of density 1.077g/ml.
To 100ml water, add 6.4g Ficoll and 10g sodium diatrizoate.
You can use the following protocol for PBMC isolation and cryopreservation.
https://www.immunetolerance.org/sites/default/files/Separation%20of%20mononuclear%20cells%20from%20whole%20blood%20using%20Ficoll%20gradients.pdf
Best.
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