[Cytometry] Propidium staining

Jordan, Robert rjordan at fhcrc.org
Wed Feb 24 12:13:29 EST 2010


We also have sometimes had variable results that we've blamed on RBC
lysis. If you don't want to use ficol we've had pretty good luck with
dextran sedimentation. Make a 10% dextran solution in PBS (Sigma
#D1037.) Then dilute blood with saline and add dextran to 1%. So--1:4:5
10% dextran:saline:whole blood. Mix and incubate at room temp for 20-30
min. Remove the upper layer containing leukocytes, spin down and
resuspend in your flow staining solution. At the end of your staining
add, ahem, 1 ml of RBC lysis solution and vortex gently. Add wash
solution, spin down, then add PI solution and analyze. There's generally
no need to incubate the RBC lysis solution for any prolonged time. It's
possible to skip this step if you can deal with the RBC contamination.
(The sedimentation isn't perfect.)

This seems like extra work, but compared to the RBC lysis time and
sometimes a second lysis the sedimentation takes about the same amount
of time.



Rob Jordan
Fred Hutchinson Cancer Research Center
D1-100
1100 Fairview Ave.
Seattle, WA 98109

rjordan at fhcrc.org
 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
> bounces at lists.purdue.edu] On Behalf Of Sasha Lazetic
> Sent: Tuesday, February 23, 2010 7:09 PM
> To: snehal mhatre; Barry Moran
> Cc: cytometry at lists.purdue.edu
> Subject: Re: [Cytometry] Propidium staining
> 
> Dear Snehal,
> 
> Your PI staining protocol looks fine. Neutrophils, however, are
terminally
> differentiated and notoriously difficult and fragile to work with.
Your
> lysing solution is more than likely disrupting the neutrophils. If
you're
> interested in the neutrophils, you may want to analyze whole blood
without
> RBC lysis, and treat the prep gingerly, no hard spins, have some
protein
> in the solution, no Ca++/Ma++, etc. Otherwise ficol separation to
> eliminate the red cells and neutrophils is the best method for
analyzing
> peripheral blood mononuclear cells (PBMC's).
> 
> -Sasha
> OMPI
> 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
> bounces at lists.purdue.edu] On Behalf Of snehal mhatre
> Sent: Tuesday, February 23, 2010 6:49 AM
> To: Barry Moran
> Cc: cytometry at lists.purdue.edu
> Subject: Re: [Cytometry] Propidium staining
> 
> Thank you so much for your suggestions.
> 
> Actually PI which i was using was prepared few months back and i think
due
> to evaporation, the solution had become concentrated. so i prepared
fresh
> PI
> solution and now it is working well for lymphocytes. but my
neutrophiles
> are
> showing 100% non-viability. I am using fresh human whole blood, lysing
> with
> ammonium chloride lysing solution without fixative and then cells are
> stained using 2uL of 0.5mg/ml PI solution.
> What could be the reason for it?
> 
> Thank you.
> 
> On Thu, Feb 11, 2010 at 3:15 PM, Barry Moran <barry.moran at tcd.ie>
wrote:
> 
> >  Hi Snehal
> >
> >
> >
> > I find PI gets into non-viable cells almost instantaneously, so you
dont
> > have to wait the few minutes or spin down again etc. I use 1ug/ml
final
> > volume of PI (not clear what conc. "2ul" is)- perhaps you're using
it
> far
> > too concentrated and background is changing. Also, best to look at
the
> > fluorescence in log scale (unlike for example when looking at cell
cycle
> > using PI, on lin scale). FL3 is probably right, though I'm not sure
what
> > cytometer you're using (channel filters vary between machines).
> >
> >
> >
> > It should be fairly straightforward then- all non-viable cells
should
> > fluoresce high (maybe 100 fold brighter) at ~620nm (probably in FL3
on
> your
> > machine); viable cells should be basically same as background.
> >
> >
> >
> > All the best- let me know if you have any further questions.
> >
> >
> >
> > Barry.
> >
> >
> >
> >
> >
> > Barry Moran
> >
> > Research Officer and Manager
> >
> > Flow Cytometry Facility
> >
> > School of Biochemistry and Immunology
> >
> > Lab 1.8, Biochemistry Building
> >
> > Trinity College Dublin
> >
> > Ireland
> >
> >
> >
> > Lab:    +353 1 896 2761
> >
> > Mob: +353 87 921 0811
> >
> >
> >
> > barry.moran at tcd.ie
> >
> >
> >
> > http://www.tcd.ie/biochemistry/flow
> >
> >
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: cytometry-bounces at lists.purdue.edu [mailto:
> > cytometry-bounces at lists.purdue.edu] On Behalf Of snehal mhatre
> > Sent: 10 February 2010 04:17
> > To: cytometry at lists.purdue.edu; cytometry at flowcyt.cyto.purdue.edu
> > Subject: [Cytometry] Propidium staining
> >
> >
> >
> > I have to stain my cell line (K562) with PI (Propidium Iodide) to
check
> the
> >
> > viability. I am adding 2uL of PI and incubating it at Room
temperature
> for
> >
> > 3mins and the aquiring on flow cytometer without washing. The peak
is
> >
> > shifting. So, i tried it with freshly isolated human PBMC but I am
> getting
> >
> > the same results.
> >
> > I even tried giving it a wash before aquiring, but it didnt made any
> > change.
> >
> >
> >
> >
> >
> > I m aquiring PI in FL3.
> >
> >
> >
> > Can anyone help me out with this? I wanted to know is my protocol is
> right
> >
> > or can anyone suggest me any changes.
> >
> > _______________________________________________
> >
> > Cytometry mailing list
> >
> > Cytometry at lists.purdue.edu
> >
> > https://lists.purdue.edu/mailman/listinfo/cytometry
> >
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