I tried to stain splenocytes with cocktail containing FITC CD3 and APC NK1.1, but did not get the feasible results. Please suggest the appropriate marker for detecting NKTs in mice splenocytes.
We sort NKT cells from mouse spleen regularly:B220, TCRB, CD1D Tetramer and NK1.1 if necessary
Gating strategy: negative B220, and double positive for TCRB and CD1D Tetramer for total NKT population then gate NKT looking for NK1.1 expression.
Hope that helps
MW
I would try looking at CD160 this is expressed on Murine NKT. So a mix of CD3, NK1.1 and CD160 should give you a proper representation of NKT cells.
Did you observe too few or too many NKT when you ran the flow?
Definitly make sure you are using an FMO (fluor minus one) when using NK1.1 it is not super robust in expression. I've attached just one of several types of CD160.
I'd agree with Matthew the best way would be Nk1.1 at the end for seperation. An lineage negative markers certainly help.
Tabasum
NKT population with mouse spleen will be "low" in the overall FACS analysis. In our sorting experiments with the strategy above the total NKT population is < 1% of total cells. We are generally happy with sorting a population of 0.5%. To save time we enrich our NKT population by 1 of 2 ways; either a depletion of B220+ cells or a positive selection of Tet+ cells. The enrichment will boost your NKT population to around 5%.
What % are you seeing
What are you activating with? One of the problems in NKT cell biology is that when activated, they downregulate the TCR/CD3. So in an unchallenged state you may see 5%, but that drops to 1% due to TCR downregulation not disappearance of your cells. I agree with Matthew that you should use CD1d tetramer.
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