Role of pH in RBC lysis solution
I want to ask aquestion in context of RBC lysis solution used in flow cytometry
We have been using our own lab made lysis solution containing ammonium chloride sodium bicarbonate and edta but when we keep the cells for more than 15-20mins in the solution out neutrophills convert into debris Or start to loose there integrity and shift to lower forward and side scatter.
Does pH play an important role here?
How long we can store the solution?
I have never kept cells in RBC or ACK lysis (1mL) buffer for longer than 1-2 minutes before quenching with PBS (9mL). You may be incubating for too long and killing your immune cells.
For RBC lysis step, I usually keep the cells (spleenocytes) in RBC lysis buffer for 10 minutes.
You should not keep more than 10 minutes. 10 minutes is enough for the lysis of red blood cells.
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