[Cytometry] CD115 staining in blood

Brian McFarlin bmcfarlin at mail.coe.uh.edu
Thu Nov 26 14:14:47 EST 2009


Similar to Adam, my group has also been using the eBioscience CD115
antibodies. Specifically, we have used the biotin version with a SA-PerCP5.5
secondary. We also recently purchased the unconjugated version and
conjugated it with a lightening link kit for APC-Cy7 (for those you you not
familiar with lightening link this is an excellent product for conjugation
and it very easy to use). We have acquired our CD115-B-SA-PerCP5.5 on a
Guava EasyCyte Mini and gotten very good resolution between positive and
negative populations. We typically dilute the stock antibody 1:200 with flow
staining buffer and then use 10 uL to stain 20 uL of mouse whole blood.

During a recent demo of the new Millipore 8ht (which is an outstanding
instrument for those of you who have not see one, not many places you can
get a true 2-laser, 6-color machine with automation for 100K USD), we did
some 6 color phenotyping of murine blood monocytes. We used the
CD115-APC-Cy7 antibody (from eBioscience with the lightening link
conjugation) and got such a strong signal (which I think is due to the high
power red laser on the 8ht along with the new Millipore high-voltage option
on the PMTs) with a 1:200 dilution that we are actually diluting the
antibody even more (still working on the titration). In these experiments,
we also stained the blood for CD11b, CD14, and GR-1. Based on these markers
CD115 is a very good marker of murine blood monocytes. It sounds like you
are interested in classical and non-classical monocytes, in order to do this
you really need to include GR-1 to separate the two populations.

We use a standard staining protocol as well. Essentially we combine 20 ul of
murine blood with an FC receptor blocking cocktail (from eBioscience), wash,
stain with diluted antibodies, wash, fix with to a final volume of 160 ul.
With this volume on the EasyCyte Mini (or 8ht) we end up with about 400-450
leukocytes/uL of final volume.

Let me know if you need any additional help,

Brian


-- 
Brian K. McFarlin, PhD
Associate Professor Exercise Physiology, Nutrition, and Immunology
University of Houston
Department of Health and Human Performance
3855 Holman St.
Houston, Texas 77204-6015
(713) 743-9929 phone
(713) 743-9860 fax



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On 11/25/09 2:00 PM, "Adam ." <anonwums1 at gmail.com> wrote:

> We've been using CD115 APC or CD115 PE from eBiosciences to do monocyte flow
> in bone both marrow and blood pretty regularly using a pretty standard RBC
> lysis protocol. Although to be honest, we've never really validated that
> these are indeed monocytes using other methods. We do know that CD115 in the
> bone marrow correlates pretty strongly with expression of CX3CR1 in the
> fractalkine GFP mice, which should be monocytes. What do you mean by "good
> differentiation of the classical monocyte population?" With the eBiosciences
> antibody, we've found that you need a pretty bright fluor to get any good
> staining and also we use a lot of antibody to get reliable staining (~1 uL /
> 10^6 cells).
> 
> Adam
> 
> On Wed, Nov 25, 2009 at 1:36 PM, Christopher Norbury <ccn1 at psu.edu> wrote:
> 
>> Folks,
>> 
>> We have been trying for what seems like aeons to get good CD115 staining of
>> mouse blood monocytes with little or no success.  We have tried a number of
>> clones, different conjugates, different methods of lysis but the staining
>> that we get does not give good differentiation of the classical monocyte
>> population.  A collaborator has suggested a number of different approaches,
>> none of which have really helped our signal.
>> 
>> Anyone got any good ideas - or is this just a sketchy antibody?
>> 
>> Chris
>> 
>> 
>> --
>> Chris Norbury, Ph.D
>> Associate Professor of Microbiology and Immunology
>> Scientific Director, Flow Cytometry Core Facility.
>> Penn State Milton S. Hershey College of Medicine
>> Room C6760, Dept. of Microbiology and Immunology, H107
>> 500 University Drive, Hershey, PA 17033-2360
>> 
>> Lab      717 531 0624
>> Office   717 531 7204
>> Fax      717 531 6522
>> Email    ccn1 at psu.edu
>> Web      http://fred.psu.edu/ds/retrieve/fred/investigator/ccn1
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