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@flowcyt.cyto.purdue.edu PH:317-494 6449 FAX:317-494 0517
web http://www.cyto.purdue.edu
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