Hi,
I am choosing antibody of CD45 for flow cytometry, my sample is mice colon tissue, but there are three types of CD45 antibody, named CD45, CD45.1 and CD45.2, Which one should I choose? Thanks！
Xin
CD45.1 and CD45.2 are allelic variants of CD45, and differ between strains (e.g. C57BL.6 and Balb/c are CD45.2, FVB are CD45.1). Not knowing what mice you're using, the safest option here is to use anti-pan CD45. Clone 30-F11 works well.
CD45.1 and CD45.2 are allelic variants of CD45, and differ between strains (e.g. C57BL.6 and Balb/c are CD45.2, FVB are CD45.1). Not knowing what mice you're using, the safest option here is to use anti-pan CD45. Clone 30-F11 works well.
Hello
I agree with Ben Roediger . Also , CD45 identification of three alleles in inbred murine strains, Ly5 a (CD45.1),Ly5 b (CD45.2), and Ly5 c . The CD45.2 allele is expressed by most of the strains, while the CD45.1 allele is found in only a few . for that use CD45 , this’s the best choice for you .
Reference : Zebedee S. L., Barritt D. S., Raschke W. C. (1991) Dev. Immunol. 1:243–254.
I just checked BioLegend website and you picked the right antibody (clone 30-F11).
Remember, most colon cells are CD45 negative epithelial cells. In the absence of inflammation a minority of cells should be positive for CD45.
Did you exclude dead cells? They might stain positive and mask the real CD45+ cells.
If you still see no positive cells, I would suspect an over-compensation problem.
Hi Eric,
Thanks for you information.
I collectted the colon inflammatory cells and lymphocyte cells for flow cytometry.
Yes, I co-stain 7AAD to exclude the dead cells, and there should be amount of CD45 positive cells, Because I also got the CD11c positive and CD11b positive cells.
I want to make sure that the pacific blue CD45 antibody is directly-conjugated from biolegend, right? maybe I need to use higher antibody concentration,
A number of possibilities with regards to the lack of detection:
1) Your pacific blue is off. Pacific blue has a limited lifespan, and if your antibody is more than 12 months old there is every chance that it has simply lost its fluorescence. Even after 6 months, one can see a drop in signal. Test the antibody using a small number of splenocytes.
2) Your settings are wrong on the flow cytometer. Specifically, your V450 PMT may be set too low. Again, you can check this by staining a small number of splenocytes with your antibody.
3) You are somehow excluding the CD45+ cells from your gating strategy. This would most likely occur as a result of over- or under-compensation, but it's possible that the signal is too high or too low, and they're not appearing in the expected area. Best to stain splenocytes with the exact same antibody cocktail at the same time as you stain your colon cells, apply the same settings and use the same compensation matrix. This will check for a number of issues, including the rarity issue flagged by Eric.
4) Your signal is "swamped out" by other fluorescent signals. Pacific blue emits in the autofluorescence range, and cells from peripheral tissues can exhibit a lot of autofluorescence. Try gating V450 vs V525 (the next channel down) to see whether you can discern the positive signal from the autofluorescence spike. Similarly, if you have DAPI in your sample, make sure you're gating UV440 vs V450, so that you can see the relative bleed-through of the DAPI and pacific blue signals.
Some final notes:
I generally use CD45 on APC-Cy7, since there is a lot less autofluorescence in the infrared range. And I always have dead cell exclusion. This is particularly important for looking at peripheral tissues, since the cell extraction process causes a lot of death.