red blood cell lysis - (Jan/28/2005 )
Does anyone know the BEST way to lyse red blood cells from mouse peripheral blood (preserved in 0.05 mM EDTA solution) for FACS analysis.
I have been using ammonium chloride buffer. Typically, this method requries that I repeat the procedure on my sample several times before I get any lysis at all. 
Dear blossom,
I am also using ammonium chloride. But it works well.
The ingredients are:
0.16 M NH4Cl
0.17 M Tris-HCl, pH 7.65
The working solution is 90ml of NH4Cl and 10ml of tris-HCl, adjust to pH 7.2
Best regards
Hadrian.Trajunas
i agree, AC works for me.
once you have the pellet
you just put in a mL of AC for two minutes and then put 9mLs of DMEM+5%FCS
and centrifuge
after that , the blood cells should have lysed
I am also using ammonium chloride. But it works well.
The ingredients are:
0.16 M NH4Cl
0.17 M Tris-HCl, pH 7.65
The working solution is 90ml of NH4Cl and 10ml of tris-HCl, adjust to pH 7.2
Best regards
Hadrian.Trajunas
I also want to lyse rbc but probelm is that after using 0.8 M NH4Cl few rbc was still there under microscope.
how one can understand that all rbc are lysed or not
For efficient RBC lysis, try sterile distilled water. Add a ml of water to your resuspended cell pellet count to 10 seconds and the top it with 15-20 ml of normal media. Be careful with the count because you don't want to lyse all the cells. Centrifuge the cells for 5 mins and resuspend pelllet in fresh media. Allow the cell debris to settle, then transfer the cells to a fresh tube. Hve been using this method for years.
how about sodium azide?
There are several commercial formulations on the market from Roche and other places which work like a charm on mouse blood.