Hi Ayham,

Unfortunately the protocols, markers, and reagents available for rat flow cytometry are less abundant, since the mouse is the immunologist's model. I'm working on developing some rat blood & spleen panels too.

I agree with Thomas that some of the CD markers do identify the exact same subsets as in mice & men, but I would caution you to review the literature carefully since there are differences.  For example, rat monocytes can and do express CD4 and/or CD8, so you can't skip using CD3 as a pan-T marker as is sometimes done when working with mouse or human samples in a Treg panel for example... For B cells CD45RA is the only flow marker I have seen used so far (still looking), and I am trying to get it to work here.  For neutrophils I am going to try using HIS48, just ordered it. For activation markers I'm looking at RT1B (MHCII) on B cells, CD25 on T cells, CD161 on monocytes. NK cells are CD161-bright, much brighter than CD161+ monocytes.

Another thing to consider if you're interested in a basic hematology panel is that in the rat, on a CD45 vs SSC plot (typical differential), there are mature erythrocytes and nucleated erythroid cells confounding the myeloid population; this doesn't happen in mouse or human.  This can be overcome by gating with a nuclear stain and CD71 Ab. See the attached paper, "Differential Analysis of Rat Bone Marrow by Flow Cytometry" by Saad et al in Comparative Haematology International (2000) 10;97-101. This differential stain is working for me on Sprague-Dawley rat blood.

Hope this helps, good luck!

Similar. But, some companies suggest special antibody cocktail for cell discrimination (such as Pharmingen).

The CD names are the same, but the actual clones will be different.

CD3 T cells

CD11b for monocytes -- you need CD43 to distinguish the monocyte subtypes -- CD43(hi) = non classical and CD43(lo) = classical

For neutrophils, there is an antibody called RP-1 which will label both neutrophils and eosinophils, which can then be distinguished using SSC (eosinophils have higher SSC than neutrophils).

Hi Ayham,

Unfortunately the protocols, markers, and reagents available for rat flow cytometry are less abundant, since the mouse is the immunologist's model. I'm working on developing some rat blood & spleen panels too.

I agree with Thomas that some of the CD markers do identify the exact same subsets as in mice & men, but I would caution you to review the literature carefully since there are differences.  For example, rat monocytes can and do express CD4 and/or CD8, so you can't skip using CD3 as a pan-T marker as is sometimes done when working with mouse or human samples in a Treg panel for example... For B cells CD45RA is the only flow marker I have seen used so far (still looking), and I am trying to get it to work here.  For neutrophils I am going to try using HIS48, just ordered it. For activation markers I'm looking at RT1B (MHCII) on B cells, CD25 on T cells, CD161 on monocytes. NK cells are CD161-bright, much brighter than CD161+ monocytes.

Another thing to consider if you're interested in a basic hematology panel is that in the rat, on a CD45 vs SSC plot (typical differential), there are mature erythrocytes and nucleated erythroid cells confounding the myeloid population; this doesn't happen in mouse or human.  This can be overcome by gating with a nuclear stain and CD71 Ab. See the attached paper, "Differential Analysis of Rat Bone Marrow by Flow Cytometry" by Saad et al in Comparative Haematology International (2000) 10;97-101. This differential stain is working for me on Sprague-Dawley rat blood.

Hope this helps, good luck!

I am really thankful for you all

I will search more and give my input for this question and also as Philippa I would like to share this paper.

Please let keep this post active by adding input for these cells

TH1

TH2

B cell

Monocyte

NK

Neutrophils

Dendritic cells

thanks

Comapred to the situation in humans and mice, the rat portfolio of anibodies is much more narrower. Hovever, particular clones have the same CD numbers (CD3, 4, 8 etc), ant they are commercially available.

Thank you all for the information above! I am still looking for a suitable antibody for (naive) B-cells for flow analyses in rats. I am not sure whether CD45RA is expressed on naive B cells or not?! Does any one of you have experience with that?

I did something following Thomas and Ursino strategy.

However, all of rat blood neutrophils expressed CD43. So CD11bCD43 staining cannot be a good strategy to identify M1 or M2 phenotype,

Moreover, there were still about ~40% CD11b/c+ cells (netrophils with high SSC-A were excluded) which cannot detected by CD161 antibody.

I did another test, which indicated that anti rat CD161 detected tissue neutrophils.

I was wondering

M1?=CD45+CD11B+CD43-

M2?=CD45+CD11B+CD43+CD4+

Monocytes?=CD45+CD11B+CD4+

Check this out:

https://www.researchgate.net/publication/288001581

Article Novel Markers to Delineate Murine M1 and M2 Macrophages

Has anyone tried RP-1 as a neutrophil marker in rat?

Is it a reliable marker for neutrophils? or any other suggestion f or a neutrophil marker in rat?

RP-1 is good for neutrophils in the rat -- probably the best you'll find. Use it it conjunction with CD11b/c.

From memory it didn't label eosinophils, but you might want to double check.

I ordered RP-1 from BD half a year ago and it did not stained efficiently with rat blood neutrophils (FCS file is existed now)... I contacted the company and sent my staining protocol, however, it still remained unsolved...anyway I do not stain rat blood neutrophils anymore...

Hi, I don't know if this is still useful , but I could identify all the immune populations in rat peripheral blood using panel given in this study : Article A Single 9-Colour Flow Cytometric Method to Characterise Maj...

I am now wondering about tissue specific marker for rat neutrophils . Let me know if someone here tried any .

Thanks