Hello, Does anyone have any experience with isolation of CD8+ cytotoxic T cells and natural killer cells from rat spleen? I'd prefer kit-based if possible, but any protocols you can provide would be helpful. I am fairly new to cell isolation, and all the kits I am coming across are mice-based. I see this kit for CD8+ T cell isolation from StemCell ( https://www.stemcell.com/easysep-rat-cd8-t-cell-isolation-kit.html) but I don't see many citations for the rat version of the kit. I can't find any kits for NK cell isolation from rat spleen.
Anything you could tell me about the process (best practices, things to avoid, successful kits and techniques) would be very helpful.
Dear Hannah,
I would suggest you to go for FACS sorting rather than Magnetic sorting if you have access to sorter.
That would be easy protocol and you need not to verify the sorted cells (in magnetic sorting you need to verify your collected cells by flowcytometry).
In flowcytometry, you need specific antibody stainig for Cytotoxic T cells (OX8) and NK cells.
Attaching a paper for negative selection of Rat NK cells sorting. By amending the method you may go for positive selection for cytotoxic T cells and NK cells from rat. Buy biotinylated antibodies for both cells fraction and sort with the help of anti-biotinylated beads in MACS.
Dear Hannah,
I would suggest you to go for FACS sorting rather than Magnetic sorting if you have access to sorter.
That would be easy protocol and you need not to verify the sorted cells (in magnetic sorting you need to verify your collected cells by flowcytometry).
In flowcytometry, you need specific antibody stainig for Cytotoxic T cells (OX8) and NK cells.
Attaching a paper for negative selection of Rat NK cells sorting. By amending the method you may go for positive selection for cytotoxic T cells and NK cells from rat. Buy biotinylated antibodies for both cells fraction and sort with the help of anti-biotinylated beads in MACS.
Jitendra Kumar Shandilya thank you for your answer! For cytotoxic t cells, would selection for CD4-/CD8+ be sufficient to collect the cytotoxic T cells? We want to then use them in a chromium-51 assay to identify cytotoxic ability. Same for the natural killer cells - CD3-/NKPR1+ to collect them?
@Mudassir Khan do the selection criteria I listed in my reply to Jitendra sound okay? I've never done FACS before so this is all new to me
Hannah, in a single tube you should add CD3,CD8,CD4 and CD16/56. On FACSAria you may collect 3-4 population (NK cells as well as Cytotoxic cells). You may gate accordingly; CD+CD8+CD4- for Cytotoxic t cells and CD3-CD56+ for NK (use respective markers of rat).
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