We use an easy protocol for platelets where we just spin out the RBCs then pellet the cells left in the Buffy Coat and it works well, I am wondering if the same can be done for Lymphocytes.
Thanks
I guess the key step is to purify the lymphocytes from whole blood.
Basically 2 ways:
1. Use lymphocyte preparation
2. Use ACK buffer to lysis red blood cells
Both of them are commercially available.
Density gradient centrifugation (Ficoll-Paque etc.) should give you a nicely lymphocyte-enriched preparation, but I don't see why collecting the buffy coat wouldn't work in a similar manner. Probably depends on what you want to see in your Western.
Ficoll density gradient centrifugation is best. Take Ficoll (15 ml for 50 ml tubes) and overlay that with diluted buffy coat (dilute in PBS 1:3 or 1:4). Centrifuge at 2000rpm for 20 min with very slow acceleration and deceleration (in total it takes around 30 mins). Collect the white ring at the interface (enriched with lymphocytes). Wash it twice with RPMI at 1200 rpm for 10 mins. This pellet can be used for Western blots. Best!
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