I have tried to sort CD8+ CXCR5+ in the ARIA but the purity of my sample is about 50%. After sorting, I stain my samples to verify purity but it is low. So, I thought about "downregulation" or "modifications" on expression of CXCR5.
Chemokine receptors definitely do modulate off the cell surface with ligand binding. Here's a link to a refernce article:
Article Mechanisms regulating chemokine receptor activity
I do not know if antibody crosslinking downregulates CxCR5, but many receptors like CD115/c-fms do.
Are you sorting in the cold with azide present in the sample buffer? That is one way to stop receptor modulation, though it can interfere with downstream uses, such as culturing or bioassaying the cells.
Try to change the volume you dilute your samples in to get a better purification.
I noticed that CXCR5 can be downregulated when cells are activated/stressed. Maybe your sorting protocol is exerting too much stress on the cells? Take into account that chemokine receptors are recycling at the membrane and temperature differences speed up / slow down that process.
When do you stain after sorting? Is the fluorochrome bleached while the antibody epitope is still occupied? You could try to use a different anti-CXCR5 clone. Good luck!
Tobias Kammann thanks for your answer!!
How dilution could improve the purification?
I have stained samples 1 hour after sorting with the same antibody (same clone, fluorochrome) because fluorochrome is photobleached during sorting. I tried to fix cells thinking about CXCR5 could be internalized; and I tried to stain cells about 12 hours after sorting. I can't see any difference really.
I use biotin CXCR5-streptavidin to stain my splenocytes.
Gary Lee Gilmore thanks for your answer and the article!!
I did not know about azide so good point. But, I'd like to sort CXCR5+ CD8+ T cells for RNA-seq and for culturing so I guess azide is not good choice for culturing.
The presence of azide during isolation - either sorting, MACS or even panning - definitely impairs proliferation of T cells.
I've done the experiment.
Gary Lee Gilmore I guess. I'm sorting in cold. Cells are recovered in tubes with suplemented-RPMI. These tubes are incubated with PBS-albumin the previous day.
Keeping your sample at 4oC is essential if azide is present in the buffer, and should help, but in my experience, there still can be inhibition. Washing the cells after sorting helps.
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