[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 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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