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moleculargeek21 08-19-2011 01:13 PM

Washing whole blood
 
My preparation of the buffy coat from whole blood is as follows.

I receive two purple top vacutainers treated with EDTA with between 2-4 ml of whole blood. I centrifuge these tubes at 2500 rpm for 10 minutes to separate the layers. I carefully pipette the plasma layer and store it at -80. I then fill two 15 ml conical tubes with cold 10 mM tris 1 mM edta and pipette the buffy coat layer with a few of the rbc’s into the vials. This is allowed to sit for 10 minutes on ice and then centrifuged for 10 minutes at 3000 rpm. The supernatant is poured off and another washing is carried out with 10ml of TE, this is centrifuged again for 10 minutes at 3000 rpm. Repeated washings are carried out until a majority of rbc’s are washed away. The pelleted buffy coat is then transferred to a cryo-vial for storage at -80

Would you suggest washing with TE or with a NaCl solution?

thanks and cheers,
-M


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