Many, many thanks to everyone who responded to my questions concerning
neutrophils. I have copied your responses below for everyone to see.
Thanks again! We'll report our results to everyone once we obtain them.
--Ross--
----------------------------------------------------------------------------
-----------------------------------------------------------
Ross E. Longley, Ph.D.
Immunology, Oncology and Screening Group Leader
Division of Biomedical Marine Research
Harbor Branch Oceanographic Inst., Inc.
5600 U.S. Highway #1, North
Fort Pierce, FL 34946
Phone (561) 465-2400, Ext 486
FAX (561) 465-1523
email: longley@hboi.edu
----------------------------------------------------------------------------
------------------------------------------------------------
On April 8, 1999, Ross wrote:
"Hello - Does anyone know of a good flow cytometry marker or antibody to
identify neutrophils in a population of human peripheral blood leukocytes?
CD16 has been suggested, but I believe this antigen is also expressed on NK
cells. Any thoughts on this would be greatly appreciated. Also, methods by
which one can isolate reasonably pure neutrophil populations?
Color me "neutrophil naive".
Ross E. Longley, Ph.D."
_______________________________-
Hi Ross,
I've been using CD16 conjugated Miltenyi beads for separation of eos. from
neuts. using a double density gradient 1.077 and 1.119 for separation from
whole
blood. I've recently been told by our resident hematologist that cd15 is
more
specific for neuts than cd16. I don't have Miltenyi experience with it yet
but
will very shortly. Obviously, you could sort these populations, but as the %
is
very low in normal hPB the recovery is better by bead than sorting. If you
want
more specific information please let me know.
Chris
Chris Groves
Millennium Pharmaceuticals Inc.
640 Memorial Drive
Cambridge MA 02139
617-679-7495
groves@mpi.com
__________________________________
WE use a combination of CD16 and side scatter - NKs have a side
scatter similar to that of lymphocytes, neutrophils have a
significantly higher side scatter.
regards
Hans Jürgen
___________________________________
Hello Ross,
I have found that one can get a relatively cleaned-up population of
granulocytes by using the standard hypaque centrifugation (e.g., Sigma
1077) of heparinized blood. The mononuclear cells occur at the
interface and the PMN are in the next layer above the RBC. They should
be washed to remove the Histopaque and then they are ready for use. If
you compare the interface layer cells with the PMN fraction, you should
have two populations highly enriched for PBM and PMN.
Cells prepared in this way typically have very low levels of
"activation" markers, e.g., the CR3, and, when loaded with reduced
dicholorofluorescein, have low levels of DCF fluorescence. Activation
with PMA causes 10-50 fold increases in DCF fluorescence whereas
stimulation with formylmethionyl peptides produces much less DCF fluor.
These cells can also be examined with membrane potential sensitive dyes
such as DiOC(6)3. Activation with PMA or f-MLP causes a loss of
fluorescence consistent with depolarization. Activated cells also have
very high levels of CR3 expression.
PMN should be separable from mononuclear cells on the basis of light
scatter. PMN have considerably higher SS signals than mononuclear cells
and could go off scale if one is using linear amplification with the
gain cranked-up. If the PMN are maxed-out, turn down the SS voltage
and/or the gain or go to log amplification.
I cannot comment on the effectiveness of CD16 labeling, but a
combination of it and light scatter should pull-out the PMN. If you
want to do functional tests on the PMN, then you might not want to use
any antibodies anyway.
Doug
--
*******************************************
* Doug Redelman, Ph.D. Sierra Cytometry *
* 3150 Susileen Drive Reno, NV 89509 *
* Phone: (702/775) 329-9772 *
* Email: redelman@concentric.net *
* CompuServe Number: 72607,155 *
*******************************************
Hi,
I have used Coulters CD16b which only labels neutrophils in humans. I also
have a method for neutrophil isolation if you still need it.
Beth Whalen
_______________________________
Polymorhoprep works well for isolating neutrophils and monocytes from
whole blood. 1-800-828-6686 (Gibco).
Please forward me any responses to your question. Thanks
Frank Radella II
________________________________
Leukogate by BDIS uses CD45 and CD
14 together one red, one green. Discriminates Lymphs monos and grans
Hope it helps
Phil Barren
___________________________________
I don't know about Flow markers, as the work I did with neutrophils was
about 4 years ago, before I got into Flow.
However, I routinely did neutrophil separation, then subsequent nitrogen
cavitation and sucrose-gradient centrification to isolate the plasma
membranes and cytoplasm. If you don't get someone responding with a more
recent method, I would be happy to dig up that protcol I used to use. Just
let me know!
email, or phone 404-639-2733.
Leigh Inge
_____________________________________
Anti CD18 seems to work very well for us. There are many available with
fluorochrome conjugates.
adipaula@welch.jhu.edu
______________________________________
Dear Dr. Ross
I can advice to use Sigma's Histopaque-1119 and Histopaque-1077 for PMN
separation, but to eliminate contaminating red blood cells, lysis stepp have
to be included.
Best whises
A. Boros
Budapest
Hungary
____________________________________
Hello Ross,
By using CD16+ and FS vs SS you can identify the neutrophils. By gating on
the high FS and SS granulocyte population and CD16+ cells you will have the
neutrophils.
To isolate neutrophils we layer whole blood over ficoll, spin.
The granulocytes and RBCs fall to the bottom of the tube.
Remove the supernatant.
Lyse the RBCs using Ammonium Chloride.
Then use mouse anti-CD49d to label the eosinophils (neuts don't express
this).
Use dynal beads coated with anti-mouse Ig to bind the CD49d+ cells, wash off
excess.
Use the magnet to remove the eosinophils, and you're left with neuts.
You can do a cytospin to check the purity.
Hope this helps you out.
-Maria.
_________________________________
CD16 is a good marker. If you gate on granulocytes in forward and side
scatter you will not have the "NK problem" as NK cells are smaller and
scatter less light. Therefore, they're in the lymphocyte population which
is quite distinctively different than the granulocyte population. Large
numbers of neutrophil enriched cell populations can be collected by
injecting thioglycolate IP in animals. Type in thioglycolate AND
neutrophils in Medline and you will get 19 references on the matter. If you
have any other questions please feel free to let me know.
Michael W. Olszowy, Ph.D.
____________________________________
We've used 16b (not 16). Works better than 66b which is supposed to be a
neutrophil marker. As far as the isolation proceedure goes we are still
working on that. The current method (dextran followed by ficoll) activates
the cells during the isolation. I'm going to be trying a new method next
week. If it works I'll let you know.
Margaret Tropea
____________________________________
Ross
--If you want "a" marker, CD16 will work. You can separate the NK cells
by scatter.
--If you can do three-color staining, use CD16/CD13/CD45. Gate on
CD45/logSSC and the neutrophils will appear on the right of that
display. If you look at the entire population from (normal) marrow, the
cells will form what looks like the Nike logo. The cells at the upper
left end are promyelocytes, myelocytes are at the lower left, and the
meta->band->mature neuts lie on the increasing slope to the right. If
you are looking at blood, you will only see the CD13+CD16+ group at the
upper right end.
--Good luck
Perran McDaniel
Cytometry Associates, Inc.
mcdaniel@cytometry.com
_______________________________
We use CD 66abce fitc from Dako Diagnostics to label neutrophils, usually
versus CD 33 pe and CD 45 .(3 color analysis.) We also use CD 16 versus CD
14
versus CD 45 in our routine panel but we have seen CD 16 negative
neutrophils
in some of our samples. The NK cells stain very bright with CD 16, and
neutrophils stain "medium bright". We have used CD 66 abce for some years
now
and have found it very useful in distinguishing different myeloid
populations.
Ernie Stapleton
Clinical Immunology Lab
St John's, Newfoundland
Canada
_______________________________
Ross, You may need to use more than one antibody to mark your
granulocytes. CD15 may be another choice and using it with CD16, they may
be a good tool. You can also look at CD16 vs SSC, the granulocytes will be
much more granular than NK cells and you might have a good separation. You
can also look at it from a different perspective using CD3, CD14, CD19 to
separate out your lymphocytes and monos. Using this mix, your granulocytes
would be unstained. I don't know how much this will help. Good Luck.
enoc@pharmigen.com
______________________________
I'm every bit as neutrophil naive, but stumbled across CD15 when screening
antibodies in nonhuman primates several years ago. This antibody is
reported to bind to all granulocytes and monocytes, macrophages & dendritic
cells. In my hands, it was extremely bright on human granulocytes, and
very dim on monos (negative on lymphs) in blood. I can send you my data
using Caltag's V1MC6 (anti-CD15) antibody as PE.
-Keith
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