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We routinely use water lysis, if you haven't tried it.
You add water for around two to three seconds vortexing continuously and
then 2, 5 or 10X PBS or Hank's (depending on how much water you have added)
again vortexing vigorously all the while, to bring it back to isotonicity.
We use it for flowcytometry, MLR's and CTL assays in murine cells. In our
hands, it seems to keep the leukocytes in better condition than Amm Cl.
You have to grit your teeth and trust it, though - it goes against all you
know, (I still cringe when I do it) but it works. You can do it more than
once if the first pass doesn't work well (i.e., if you chicken out and add
the concentrated buffer too soon to bring it back to isotonicity), but with
practice you can get it to work the first time always, thus minimising the
stress on the cells.
Hope this helps,
Nick
>Can anybody help with a recipe for a RBC lysis solution that I can
>use in conjunction with surface Kappa and Lambda staining. We want to
>lyse, wash, stain and then fix cells. Currently we use ammonium
>chloride with mixed sucess, we lose scatter sometimes and sometimes
>the RBC take an age to lyse. We use 2ml lysis per 100ul cells.
>Joy Mundy
>Dept. Human Immunolgy
>Institute of Medical & Veterinary Science
>Adelaide, South Australia 5000
>Ph 61-8-2287476; Fax 61-8-2324092
>jmundy@immuno.imvs.sa.gov.au
Nicholas J.C. King
M.B.Ch.B., Ph.D.
Senior Lecturer
Dept of Pathology
University of Sydney
N.S.W. 2006
Australia
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