Hi there! I’m having trouble performing red cell lysis buffers. As well as killing red blood cells the buffer also seems to be killing the lymphocytes (Isolated from human intestinal tissue). My protocol at the moment is 1:10 solution of RC lysis buffer, add to the cell solution and leave for 6 minutes at room temperature. Wash with PBS at 1500 rpm for 5 minutes. Could anyone give me any insight as to why the RC lysis buffer would be killing my entire cell pellet? Thank you!
6 min seems too long, that's why you may be getting other cells lyse as well.
What I do is use the following lysis buffer.
Ammonium chloride: 4.01g
NaHCO3: 0.42 g
EDTA(Na2): 18 mg
H2O: 500 ml
I resuspend cells in 5 ml of this lysis buffer and keep at RT for 1-3 min and then quickly add 15-30 ml serum free media or PBS and centrifuge.
After centrifugation you should be able to get white cell pellet, which means that the RBC lysis was complete.
Note: The lysis buffer works well with relatively new Ammonium chloride. If is is and old stock it might not work well in lysing RBCs.
Good luck,
S
Note: You may be able to dissolve EDTA directly so you can add required amount from a 0.5 M solution of EDTA.
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