What is the best way to isolate PBMC from blood for freezing? RBC lysis method with ammonium chloride or density gradient with Ficoll? Has anyone done comparison of both methods with cells that are frozen down for long periods of time?
Hello Helen Wong
RBC lysis method with ammonium chloride is the preferred method because it is more convenient. Lysis is much quicker than gradient separation and in general leaves the remaining white populations relatively unperturbed. Further, the yield of leukocytes from blood by lysis of erythrocytes is much higher than the yields from density gradient separation and the viability of white blood cells subjected to this treatment is also good.
Though the RBC lysis method with ammonium chloride has many advantages, density gradient with Ficoll should be used when it comes to isolating PBMCs from blood for freezing because it will provide you with purified cell population rather than just simple removal of erythroid contaminants.
Using density gradient with Ficoll, four layers will form, each containing different cell types. The uppermost layer will contain plasma, which can be removed by pipetting. The second layer will contain PBMCs and is a characteristically white and cloudy “blanket.” These cells can be gently removed using a Pasteur pipette and added to warm medium or PBS to wash off any remaining platelets. Density gradient separation may not yield as many cells as the simple lysis method, but should be used when you need to freeze PBMCs from whole blood.
Best.
Hi Helen
In my lab we have been working with Ficoll. Then you have monocytes and lymphocytes together. Is this the way it should be. I have no experience with this, but I can say that we then isolated the monocytes and these could be frozen at -80 degrees Celsius. These then remain functional for months (3-5 months). Our freezing medium 6-7 million cells/freezing medium. Medium 15% FCS +10% DMSO in RPMI1640
greetings
Jochem
We use ficoll an freeze after separation with cryocell for long time in -80.
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