Hi!
I have to carry out a mouse B and T cell immune profiling FACS based assay from animals that have been immunized as a part of my study. I am new to immune profiling and I would be happy to get any pointers on building the antibody/ marker panel and the gating strategies would be very helpful. We have a BD facs verse.
I would first think about what subsets of B/T cells you would expect in your samples. For a general immune response in mice I would suggest CD44, CD62L, ICOS, PD-1 and CXCR5 for your T cells, and CD138 and GL7 for your B cells. These are good activation markers but you can definitely add others that you are specifically looking at. Once you narrow down the targets that you would like to look for, you need to see how many color channels your flow cytometry machine can handle. Once this is done, you can order your antibodies. There are good protocols out there for the extraction and staining of your cells. Once the data is collected, you gate on your live cells by gating those that are PI/DAPI negative adn then your T/B Cells (I usually use B220 to get to B cells and CD4 and CD8 to get to these 2 populations of T cells). You can then use these populations as your base and see what other markers they are expressing. In T cells, CD44+, CD62L- are effector cells, CD44+, CD62L+ are memory cells and CD44-, CD62L+ T cells are naive. PD-1 is a good marker of exhaustion, CXCR5+, PD-1+ T cells are T follicular helper cells and ICOS is a general T cell activation marker. In the B cells, GL7 is a marker of germinal center B cells and CD138 is a marker of plasma cells. Here is a good link to describe B cell activation markers https://www.bio-rad-antibodies.com/b-cell-function-activation-lineage-marker-antibody.html. Here is another for T cell markershttps://www.bio-rad-antibodies.com/t-cell-structure-function-activation-lineage-marker-antibody.html. I hope that this helps out a little bit. If you have any specific questions I can try to answer those. Good luck!
Dear Swetha,
As John Wilson just wrote, it all depends on what are you looking for.
Here are some more suggestions on where to look for gating strategies and markers:
- The OMIPs (although most are human) provide not only with the markers but the gating strategies and reagents : https://onlinelibrary.wiley.com/doi/toc/10.1002/(ISSN)1552-4930.OMIPscollection
- The EJI guidelines can provide you with the isolation protocol and gating strategies and also help with everything that is related with Flow cytometry : Article Guidelines for the use of flow cytometry and cell sorting in...
- Some antibody companies like Miltenyi also have some guidelines on which markers to use for each populations:
https://www.miltenyibiotec.com/DE-en/resources/macs-handbook/mouse-cells-and-organs/mouse-cell-types/b-cells-mouse.html
https://www.miltenyibiotec.com/DE-en/resources/macs-handbook/mouse-cells-and-organs/mouse-cell-types/pan-t-cells-mouse.html
I hope this helps.
Good Luck
Dear John Wilson and Joana Barros-Martins both of your suggestions and references have been very useful. Thank you! I am using these along with the BD FACs reference guides to sort out the flurochromes that I might require to avoid the overlapping signals.
Swetha Raghavan I am glad that these helped! Please let me know if you have any more specific questions as you proceed with this and I will see if I can help answer them. Good luck with the experiment!
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