For those interested, here is a summary of the responses the question I posted
about red blood cell lysis and annexin binding. Thanks to all of you who
replied:
1) the use of FACS lyse (BD) was suggested. FACS lyse fixes the cell as well as
lysing them so I have chosen to use PharmLyse (also BD) which only lyses the
cells but FACS lyse also works well (see Hodge ref below) if you want to fix
your cells.
2) there was concern about being able to measure apoptosis at all in whole blood
culture. I have measured this before using the comet assay (Mut. Res. in press)
and others have measured as much as 20% apoptosis in whole blood.
3) centrifuging the blood was suggested and using the buffy coat. Since I am
trying to separate the blood after the apoptosis is expressed, I was worried
about losing apoptotic cells during centrifugation due to their change in
density.
4) ammonium chloride seemed to be the lysing agent of choice. This is the basis
of PharmLyse as well.
- A suggested method from Paula Fukushima from NIH:
"We lyse for 10 minutes without doing anything to the blood except mixing
gently. Lyse a whole tube in a 50ml conical- say 5-10cc with 40-45ml
ammonium chloride. At exactly 10 minutes, centrifuge at 1200RPM for 7-10
minutes. Empty the tube and with a Pasteur pipette gently squish to try and
get as many of the left over RBC out of the tube. Do not vortex from now
on. Take a wide-mouthed Pasteur pipette and re-suspend the pellet in a little
PBS. Add PBS to the 50ml mark and spin. Do this a total of 3 times. The
lysed and washed cells can sit for a while at RT. We have also had success
taking blood and adding RPMI to it in a 50ml conical tube and then
incubating the whole thing overnight if the specimen comes too late in the
day. "
5) the importance of having the proper Ca++ was stressed by many people for
proper Annexin binding. Resuspending the cells in 1x binding buffer (diluted in
DI water) was suggested to ensure proper Ca++ concentration.
- Greg Hodge from North Adelaide stated
"I have used FACSLyse from BD on whole blood cultures (1:2 with RPMI) and
stained with Annexin V without compromising lymphocyte viability (Hodge et
al. Increased levels of apoptosis of leukocyte subsets in cultured PBMCs
compared to whole blood as shown by Annexin V staining: relevance to
cytokine production. Cytokine 2000, 12, 1763-1768). Are you keeping plenty
of Ca++ in the staining mixture."
-Annexin labelling method of Matthew Morrow from NIH
"Normally, I prepare my annexin reaction as a "master-mix"
composed of: 100ul 1x binding buffer, 5 ul annexin, 2 ul PI (50 ug/ml stock)
for EACH tube in my assay. After washing the lyse off of the cells with PBS,
I decant as much PBS as possible and re-suspend the cells in 100 ul of the
above mix.Lastly, if you are using a commercial binding buffer
(i.e. Pharmingen), dilute the binding buffer with DI water, not PBS."
6) poor annexin labelling may be due to platelets taking up the annexin rather
than erythrocytes;
- A comment from Francis Andrews from Liverpool:
"The answer to the poor annexin labelling may be due to platelets
rather than as you suggest erythrocytes.
There are several references to platelets strongly binding annexin v. I've
been labelling PMNCs with annexin in increasing amounts of apoptosis
inducing agents, yet the annexin labelling is very uneven when compared
with the numbers of apoptotic cells seen on a light scatterplot. When I've
examined PMNCs microscopically, I've still got a lot of platelets so I'll
just centrifuge at low speed to get rid of them next time. However, this
may be more difficult to achieve with whole blood lysis. Would increasing
the amount of annexin v help? I agree with Greg Hodge that the Calcium is
critical, as I've found out myself."
7) Water lysis was not a common method used for human red blood cell lysis. One
responder mentioned using it on bovine cells but no one else mentioned using it
on human blood.
Hope this is helpful to anyone interested in this technique. We are still
working out some details but feel we are well on the way to success with the
PharmLyse and Annexin binding. Thanks again to everyone who responded to my
question.
Ruth Wilkins, Ph.D.
Consumer and Clinical Radiation Protection Bureau
Health Canada
775 Brookfield Road, PL 6303B
Ottawa, ON K1A 1C1
(613) 941-7263
FAX (613) 941-1734
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