Re: [NH4Cl lysing of murine whole blood]

From: Rana Nagarkatti (rana_nagarkatti@usa.net)
Date: Fri Nov 17 2000 - 14:47:22 EST


Hi,
 I dont know about the murine RBC lysis but for the human blood we use
bufered ammonium chloride solution. hope these suggestions help.
 1. the blood has to be at RT at 4 the membrane is stabilised and so lysis
will not be complete.
2. we take about 2 ml blood adn dilute it to 50ml with 1x buffer.
3. mix the two gently adn then incubate at 37 for 10 min.
the composition of the buffer is-
  nh4cl  32.08gm
  nah2co3  3.36 gm
  edta na2  1.41gm
 make up to 400ml with milliQ water. filtersterilize if required.
this is 10x solution. can be stored at 4 for 6 months. make up to 1x
immediately before using.
4. check the viability of the cells with trypan blue ater the spins. Generally
we go for 1000 to 1200 rpm on a bench top clinical centrifuge. also u can try
underlaying with fcs and then spinning,
hope these help
 Regards
Rana.




"Haugan, Anita" <anita.haugan@folkehelsa.no> wrote:

To all of you who use ammonium chloride lysing of RBC in whole blood samples
from mice for surface markers:

I need some advice concerning lysing of murine RBC. I have just started to
work with whole blood from BALB/c mice, and are trying to lyse the RBC with
a NH4Cl-solution. My problem is that my samples aren't completely lysed, and
one can see that the pellets are redish in some cases. My qustions are:

What is the optimal g-value for centrifugation of the samples to get maximum
lysis? We use 1200rpm (270g) for 10 min.

Is there anything else we should be aware of in using NH4Cl lysing - both
concerning the lysing method and analyzing the samples on the flowcytometer?

So if anyone have some answers/advices or best of all a detailed protocol of
how to lyse RBC in murine whole blood they could send me, I will be extremly
happy!

Thanks in advance!

Anita Haugan
Department of Vaccinology
National Institute of Public Health, Norway


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