I need to stimulate NKT cells for the measurement of IFN-gamma release from these cells by flow cytometry. Can I use a stimulant other than alpha D-galcer to stimulate these cells for this purpose?
Hi,
You can use PMA+Ionomycin and incubate for 4 hours. Do not forget to add golgi stop.
You should have one control tube +golgi stop as unstimulated.
Dear Sidiq
You may please download this paper foe assistance:.
Samuel Victor Nuvor, Hilton Whittle, Sarah Rowland-Jones, Assan Jaye. (2012) Higher expansion of IFN-g - CD4+NKT cells in asymptomatic HIV-1 than in HIV-2 individuals. World Journal of AIDS, 2:103-108
- in vivo: 1ug aCD3eAb (clone 145.2C11) i.v. for 90min; or 40ug LPS i.v. for 6h
- in vitro: PMA/ionomycin for 4h (+Golgi-Plug/Stop); or plate bound aTCRb or aCD3e-Ab
But then again, why not aGalCer? Works like a charm ;-)
Good luck, G.
Thank you all.
Can I use PMA/Ionomycin for the stimulation of NK1.1 cells too along with NKT cells for measuring the IFN-gamma release from these cells.?
if you mean NK cells with NK1.1+ cells, then PMA/Iono will stimulate those too
Hi Tabasum,
Just ensure you are starting with a pure population of NKT cells. The problem with PMA/iono or CD3/CD28 ligation is that they are non-specific. Hence if you have other population of cells in culture, they will get stimulated as well. The only way to specifically stimulate NKT cells is to use glycolipids such as alpha-galcer. I hope this helps.
Also, note that NKT cells might down regulate their TCR following stimulation.
Simon: stimulation in a more broad manner (PMA/Iono, aCD3eAb) is fine in this case as one can stain the iNKT cells specific (CD1d/aGC-tetramers). In the 4-5h time window the down-regulation is only a problem after aGalCer in vivo (that will happen in 2-3h).
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