From: Martin Poirier (coch@myrealbox.com)
Date: Wed Sep 24 2003 - 18:16:09 EST
Ok I'll summarize replies I got so far below. For additional information, know that we
collect blood in EDTA tubes, we stain 100ul of blood for approx
30 minutes, after which we lyse the RBC with cmommercial (BD) erythrolyse solution, wash
twice and fix the cells. All wash steps are
done using PBS alone.
I included the real dot plot (not my drawing) this time. Also, cells in this region comes
up HIS48+ / CD45+ and CD11b/c+. I will look with other markers tomorrow.
Thanks
Martin
Replies I got so far:
--------------------------------
Oddly enough, just today I was looking at some bone marrow aspirate cells (ficoll,
supposed CD138 negative MACS selected) and saw just such a population.
Gotten any responses yet?
--------------------------------
Beats the hell out of me. I would guess some really big lymphocytes, but I have not seen
this in RAT blood ( lysed). I have seen a separate population like this in human blood(
but not so far removed from the lymphs) when using FACS lyse. ( this reagent will
sometimes split lymphocytes into two regions on FSC with the same SSC signal
Why not stain with lymphoid , monocyte markers etc and see what you have.
Alternatively a CD14 vs CD45 "Leukogate" stained sample should let you know if they have
the same staining characteristics as lymph's, monos or grans
Let us know
Tks
Pb
--------------------------------
I have seen air bubbles (when SOMEONE runs out of sample...) come up in that region, but
I don't remember a distinct population of cells, at least in human PBMC and BMMC
isolates.
Good question.
Cris Bare
Cornell Med, NY
--------------------------------
Could it be air that is in your solution?
Ruedi
__________________________
Dr. Ruedi Braun
Senior Scientist
Department of Surgery
CSC H4/383
600 Highland Av.
Madison, WI 53792
phone: (608) 265-0500
fax: (608) 263-0547
e-mail: braun@surgery.wisc.edu
--------------------------------
Martin,
We see a similar population looking at rat BAL samples (high FSC (like
Grans/Macs)) and low SSC (like Lymphs)). For our BAL experiments we've been
staining with clone 1C7 ("Mononuclear Phagocyte" to quote the BD/Pharmingen
chart) and clone RP-1 ("Granulocytes"). In our BAL samples, about 90% of
these cells are 1C7 positive and the remaining 10% are double negative.
Greg Perry
Creighton University
Flow Cytometry Core Facility
--------------------------------
Seen this lots with all kinds of PBMC preps. They've been lymphs in my
hands (based on 3/4/45 staining).
Cheers,
Nate
Nate Regimbal
IDEC Pharmaceuticals Corporation
Cell Biology Group
Ex. 8293
--------------------------------
I guess you do some type of fixative shrinking lyse and these just did
not lyse. They should however antibody label okay
Regards
Gerhard
--------------------------------
Determining original message timestamp ..... Done.
Timestamp = 24-09-03 08:00
Retrieving original message sent by Ernest Stapleton ..... Done.
Original message:
>You don't mention how the blood is processed, washed, lysed etc. These procedures can
>cause many artifacts.
>
>Ernest Stapleton
>Division Manager
>Immunology and Genetics Laboratories
>Room 1524 Health Sciences Center
>St. John's, Newfoundland
>A1B 3V6 Canada
>709-777-8654
>
>Martin Poirier wrote:
>
>-What is this population?
>
>
>Hi everyone,
>
>I'm doing basic CD3/4/8a/45RA/161a staining on rat blood, and as far as
>I remember, I have always seen a population with High FSC/Low SSC on the
>light scatter dot plot. I wonder what this population might be as I was
>told many different things like LGL, stem cells, atypic lymphocytes, and
>I don't know who to believe
>
>I cannot find any info on the internet regarting that population
>(exhaustive info about light scatter properties of cells on FSC/SSC dot
>plots is hard to come by).
>
>I drew a picture to show the approximate appearance of the dot plots I
>see. Note that my drawing is in no way an exact representation of what I
>see. Unfortunately, I forgot the real data picture at my office, and I
>didn't want to wait until Monday to get an answer to this, hence the
>reason why I drew the mockup plot myself. The population I'm talking
>about is circled in red on my drawing, and is in the 800 range on the
>FSC axis.
>
>I see this with Sprague-Dawley (8 to 10 weeks old) and Fisher (6 to 12
>weeks old) rats. Didn't try other strains.
>
>Any input is welcome,
>Martin
>
>
>
>
>
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