NK1.1+ CD3- is enough to say NK cells.

Intracellular staining for Perforin and granzyme may be performed directly but for cytokine you need to stimulate PBMCs with PMA/Ionomycin and Brefeldin A.

For functional assay you may stimulate PBMCs with PMA/Ionomycin and Brefeldin A and CD107a antibody, followed by staining for NK.

Hii,

In terms of markers for NK cells, it may depends a bit on the organ you are studying. You can definitely do a CD45+CD3-NK1.1 general gating for your NK cells but you will need to check that there is no expression in your population for other cell types (CD19, CD11b/c, FceR1, F4/80) or alternatively you can use a Lineage staining including all those markers in the same channel to gate on the negative population for identification of your NK cells.

Additionaly, NK1.1+ includes not only NK cells but also ILC1s, to differentiate between both you could stain for CD49a and CD49b, as NK cells are CD49b+ and ILC1s are CD49a+. You could also use the transcription factors Tbet and Eomes, although this will require fixation and permeabilization of the cells.

For cytokine detection (IFNg/TNFa) you can stimulate ex vivo with PMA/Ionomycin incubated together with Brefeldine to avoid secretion and allow you to detect cytokines inside the cell (this also require fix/perm). You can also do the same for perforin and Grzn B, which should increase after activation. To test degranulation capacity, you stimulate with the same things in the presence of CD107a.

Good luck!

Hi,

The attached article is god for you.

Article Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Dear Anna, please follow an attached link.