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Red Blood Cell (Hypotonic) Lysis Buffer

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Bert Gold

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Jun 23, 1998, 3:00:00 AM6/23/98
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Colleagues,

For a DNA extraction at work, it would be good to be able to mix up
a hypotonic buffer capable of lysing red cells, but keeping white
cells (or at least nuclei) intact. Anybody know a recipie for
such a salt-water mix?

Bert Gold
Sherman Oaks, California
http://www.ktb.net/~bgold

Warren Gallin

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Jun 24, 1998, 3:00:00 AM6/24/98
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In Article <35904C...@ktb.net>, Bert Gold <bg...@ktb.net> wrote:
>Colleagues,
>
>For a DNA extraction at work, it would be good to be able to mix up
>a hypotonic buffer capable of lysing red cells, but keeping white
>cells (or at least nuclei) intact. Anybody know a recipie for
>such a salt-water mix?

When making hybridomas it is desirable to lyse the RBCs and leave the
lymphocytes viable. We resuspend the cell pellet from a single spleen in 5
ml of ice cold 0.75% ammonium chloride, on ice, five minutes, then pellet
the cells, discard the now red supernatant, and resuspend the cell pellet in
normal medium, or buffered saline. Virtually all the RBCs are gone and the
lymphocytes are still viable.
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton, Alberta T6G 2E9
Canada
wga...@gpu.srv.ualberta.ca

Simon Mauch

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Jun 24, 1998, 3:00:00 AM6/24/98
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Hi Bert,
My protocol for lysis of red blood cells:
Pellet the the blood cells and resuspend in a Tris-NH4Cl solution (0.1
ml packed cells/ml Tris-NH4Cl). Hold at room temperature for 5 to 10
min.
Centrifuge at 300xg for 10 min. (You might underlay the cell
suspension with FCS before centrifugation)
Repeat the process if red blood cells are evident in the pellet.
Wash the pellet with PBS.
Use the Pellet for DNA isolation

stock solutions:
0,16 M NH4Cl (8.3 g/l)
0,17 M Tris-HCl, pH 7.6
working solution: 9 parts NH4Cl stock solution and 1 part
Tris-solution. Adjust the pH to 7,2 with HCl

Simon


>Colleagues,
>
>For a DNA extraction at work, it would be good to be able to mix up
>a hypotonic buffer capable of lysing red cells, but keeping white
>cells (or at least nuclei) intact. Anybody know a recipie for
>such a salt-water mix?
>

Otto Zach

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Jul 1, 1998, 3:00:00 AM7/1/98
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For routinely RNA extraction out of 5ml whole blood I use the following
protocol:
First of all remove plasma, then add 5ml of 0.15M NH4Cl (8g/l), 0.01M KHCO3
(1g/l) (precooled to 4°C) and shake for 5min, centrifuge for 10min at 4°C
with 2.000g and pour away supernatant. Repeat steps if necessary and
dissolve cells in lysis buffer (e.g. TRIzol). Good luck!

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