[Cytometry] flow cytometry of mouse BAL fluid

Oliver Dienz odienz at uvm.edu
Sat Jan 30 00:16:03 EST 2010


Hello Ulf,

Sorry for the late reply. I did stain BAL cells from influenza  
infected mice a few times and can share some of my experiences with  
you. The panel I used was mostly determined by the antibodies I could  
get my hands on but for the most part it worked quite well. The  
samples  were run on a BD LSR2 with lasers at 355 nm, 405 nm, 488 nm,  
and 633 nm. The panel was:

UV-Live/Dead (Invitrogen)
CD4-PacificBlue
GR1-PacificOrange (good fluorophore for this usually bright marker)
CD11b-Alexa488
CD11c-PE (or MHC2-PE)
CD8-PE-TexasRed
F4/80-PE-Cy5.5 (recommended from our lung specialist as best marker  
for alveolar macrophages)
7/4-Alexa647 (see below)
B220-APC-Cy7
TCRbeta-PE-Cy7

The 7/4-neutrophil marker was recommended by a collaborating group and  
is available from AbD Serotec; unfortunately only in a limited number  
of colors. It's very bright in Alexa647 and requires careful titration  
and setting of voltages e. g. with thioglycollate induced peritoneal  
neutrophils. According to our collaborators, neutrophils are F4/80neg  
and 7/4-high but the percentage of that group is quite a bit lower  
than what we get by a simple Giemsa stain of BAL cells so I have some  
lingering doubts about that. Sorting those populations and checking  
them in a microscope is still on my to-do list. When you run your  
samples on an Aria anyway it may be a good idea to confirm your flow  
data with the cell morphology.
The use of an apoptosis stain had been quite useful for "cleaning up"  
the stain a bit but may not be absolutely required. However, at least  
in our case of an acute infection you will not see the nice, distinct  
populations you usually see in the spleen. That is the reason why I  
try to hit most cell types with two markers (e. g. CD4 and TCRbeta or  
F4/80 and CD11b) to be able to clearly identify each population. We do  
not see eos in our BAL so I did not look for them.

Good luck,

Oliver

Quoting Ulf Gehrmann <ulf.gehrmann at ki.se>:

> Dear all,
> I am interested in analysing and quantifying cells of mouse BALf. I
> would like to set up a panel distinguishing between macrophages,
> neutrophils, lymphocytes and eosinophils while running counting beads
> to approximate the cell number.
> Does anyone have experiences with mouse BALf and can you recommend a
> combination of antibodies?
> I appreciate any help or comments regarding this matter.
> Best wishes,
> Ulf Gehrmann
>
> Ulf Gehrmann, PhD-student
> Karolinska Institutet
> Department of Medicine, Solna
> Clinical Allergy Research Unit
> Karolinska Universitry Hospital Solna, L2:04
> 17176 Stockholm
> Sweden
>
> tel.:0046-8-51776696
> mobil:0046-704-351136
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
>



-- 
Oliver Dienz
University of Vermont
Department of Medicine/Immunobiology program
Given Bldg. D305
89 Beaumont Ave.
Burlington, VT 05405
USA
office: 001-802-656-8180
lab: 001-802-656-1018
fax: 001-802-656-3854
email: odienz at uvm.edu



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