I have used the following markers in a flow cytometry panel to assess mouse samples from bone marrow, spleen, fat pad, peyer’s patch and lymph node:
Ly6G
F4/80
CD11b
CD11c
SiglecF
FceR1a
I selected the granulocyte population in the fsc-a vs ssc-a plot. Then I gated on the viable cells and then on the singlets. To identify neutrophils I gated on fsc-a and Ly6G. Then I took the neutrophil population and divided it between siglecf and Ly6G, and labeled the double positives eosinophils. Is this correct? If so, can I use the same strategy for all the organs?
I also divided the granulocyte population between cd11c and cd11b. I then took the cd11c- and cd11b+ population and divided it between cd11b+ and f4/80. Is this the right way to gate on macrophages?
What else can I do with these markers using what gating?
Thank you!
Hi Luisa,
I just briefly wanted to email you concerning your FACS gating question. Please check the attached paper from the Ziegler group. In the supplement you find a gating strategy for eosinophils, alveolar & interstitial macrophages & neutrophils (in the lung). Depending on your experimental conditions you may or may not have a lot of alternatively activated macrophages that may be somewhat similar to the alveolar ones described in the gating strategy below.
http://www.jimmunol.org/content/early/2012/12/28/jimmunol.1201808
http://www.jimmunol.org/content/jimmunol/suppl/2013/01/04/jimmunol.1201808.DC1/12-01808_S1-3.pdf
The FceR1a marker is generally considered for the identification of mast cells or basophils. For more details I'd suggest another paper that outlines flow cytometry identification for murine immune cells under homeostatic and inflamed conditions.
Article A Protocol for the Comprehensive Flow Cytometric Analysis of...
I hope that helps with your analysis.
All the best & good luck for your experiment,
Michael
Hi Luisa,
I just briefly wanted to email you concerning your FACS gating question. Please check the attached paper from the Ziegler group. In the supplement you find a gating strategy for eosinophils, alveolar & interstitial macrophages & neutrophils (in the lung). Depending on your experimental conditions you may or may not have a lot of alternatively activated macrophages that may be somewhat similar to the alveolar ones described in the gating strategy below.
http://www.jimmunol.org/content/early/2012/12/28/jimmunol.1201808
http://www.jimmunol.org/content/jimmunol/suppl/2013/01/04/jimmunol.1201808.DC1/12-01808_S1-3.pdf
The FceR1a marker is generally considered for the identification of mast cells or basophils. For more details I'd suggest another paper that outlines flow cytometry identification for murine immune cells under homeostatic and inflamed conditions.
Article A Protocol for the Comprehensive Flow Cytometric Analysis of...
I hope that helps with your analysis.
All the best & good luck for your experiment,
Michael
Basically look at what Michael here suggested. Those are great references for what you are trying to do. Also, what is you goal? Are you looking for other cells? Do you want to have a panel that includes exactly what? (That would help us giving you better answers). CD11b can be a tricky marker as its surface expression levels vary a lot depending on the cell type, anatomical location and activation state, and it is expressed in many cell types. CD11b+ F4/80+ should give you though a clear mature macrophage population. You might also want ot use an FC-block reagent before staining to avoid unspecific signals, especially when looking at myeloid cells and such.
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