Lysing Solution - Again...Formula

PAUL@flowcyt.cyto.purdue.edu PAUL at flowcyt.cyto.purdue.edu
Mon Feb 10 17:51:23 EST 1997


Colleagues: Several months ago there was considerable discussion 
regarding lysing solutions. Sine this is probably one of the most 
frequently used reagents eveyone should know lots about it!

I have encountered a small problem in finalizing the reagents for 
Current Protocols in Cytometry which is near publication. I proposed 
to place common reagents into the appendix, one of which is the 
following formula for "Lysing Solution". However, one author has 
refused to accept this as correct and I need to seek the wisdom 
of the great cytometry minds of the outernet!

Question 1: Can you make up 10 x conc and store it as we propose?
    (my answer: well I've done it this way for 15 years - but somehow 
that's not a very scientific explanation.....)

Question 2: Can you pH this solution and if so what do you use? 
(pH comes out at about 7.2 anyway-remembering these are carbonate 
buffers)

Question 3: Is there any preference for disodium or tetra sodium 
EDTA (each at an appropriate concentration).
    (my answer: both seem to work fine at the correct conc)

The proposed final formula is below. I would appreciate comments. I 
have about 2 days to make a decision - if I make the wrong one, 
cytometrey may for ever be changed ..(."my lysing solution doesn't 
work anymore...."!!). Thanks for the advice.

ans if anyone knows what the ORIGINAL REFERENCE for this is, I'd like 
to know.

Paul Robinson
---------------------------------------------
Ammonium chloride lysing solution, 10x

g NH4Cl (1.5 M) (final (.15M)
g NaHCO3 (100 mM) (final 10 nm)
g disodium EDTA (10 mM) (final 1 mM)
Q.S. to 1 liter with H2O; pH will be approximately 7.2
store 6 months at 4degC

Working solution:  Dilute 1:10 with water to make fresh working 
lysing solution just before use. Keep working solution cold and 
discard any unused portion.

This solution is used to lyse erythrocytes. Never store lysing 
solution at <10x concentration, as it will form ammonium carbonate, 
which will not lyse erythrocytes.
------------------------------------------------------------------

J.Paul Robinson, Purdue University Cytometry Labs
Professor of Immunopharmacology
robinson at flowcyt.cyto.purdue.edu PH:317-494 6449 FAX:317-494 0517
web http://www.cyto.purdue.edu



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