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MacFarlane AW , Oesterling JF , Campbell KS
Measuring intracellular calcium signaling in murine NK cells by flow cytometry
Methods Mol Biol. 2010 ;612 :149-57
PMID: 20033639    PMCID: PMC2798144   
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Abstract
This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK cells using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller NK cell subpopulations can be measured simultaneously from a mixture of freshly prepared total splenocytes without resorting to prior cell sorting or expansion in culture. Briefly, cells are harvested and stained to mark the populations of interest, then loaded with indo-1 AM dye and analyzed on the flow cytometer. After an appropriate baseline is established, the cells are treated with a biotinylated antibody to activating receptors, which are subsequently cross-linked by addition of streptavidin. The increase in intracellular calcium is quantified by measuring a shift in the indo-1 emission spectrum that takes place when the dye becomes bound to calcium.
Notes
MacFarlane, Alexander W 4th Oesterling, James F Campbell, Kerry S CA06927/CA/NCI NIH HHS/United States R01-CA083859/CA/NCI NIH HHS/United States R01-CA100226/CA/NCI NIH HHS/United States T32-AI007492/AI/NIAID NIH HHS/United States Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't United States Methods in molecular biology (Clifton, N.J.) Nihms152769 Methods Mol Biol. 2010;612:149-57.