Hi,
I am trying to stain human PBMC (population of interest is NK cells) for ribosomal pS6 and I go according to below mentioned steps but I do not get proper pS6 detection and also the surface staining looks very bad. I believe it is the fixation process that affects the surface staining, but not sure how to overcome this issue.
1. Thawing samples followed by staining for live/dead marker (Fixable Viable Stain) 20 min 37C
2. Spin down 300g 5min 20C and remove the supernatant
3. Overnight with different cytokines (resting as a control)
4. Spin down 300g 5min 20C and remove the supernatant
5. Fix the cells using pre-warmed BD Cytofix buffer 12min 37C
6. Spin down 300g 5min 20C and remove the supernatant
7. Stain for surface markers including CD3, CD56, NKG2A and KIRs at room temp 30min
8. Spin down 300g 5min 20C and remove the supernatant
9. Permeabilise the cells using BD Perm Buffer III on ice 30min
10. Spin down 300g 5min 4C and remove the supernatant X2
11. Stain for pS6 using BD AF488 antibody RT 60min in the dark -stain buffer used for prepping
12. Spin down 300g 5min 20C and remove the supernatant
13. Re-suspend cells in BD stain buffer prior to acquiring
Any feedback is highly appreciated.
Thank you
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