This protocol was used in the above Nature Immunology paper.
Method Article
Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+
https://doi.org/10.1038/nprot.2007.66
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 29 Jan, 2007
You are reading this latest protocol version
This protocol was used in the above Nature Immunology paper.
Serum (for RPMI with 2% serum, if cells require serum).
PREPARATION OF INDO-1:
Store excess RPMI-diluted 10 mM Indo-1 at 4°C. In our laboratory, the 10 mM Indo-1 solution has been tested for stability up to 24 hrs.
PREPARATION OF IONOMYCIN:
Incubate cells (2x107/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading.
Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument.
Wash cells twice in RPMI and suspend them at the desired concentration (usually 2x106/ml). Higher cell concentrations (4x106/ml) are required when the cells of interest represent less than 10% of the total population. Cells can be kept at 20° to 25 C unless the antigen is subject to capping, otherwise use 4° to 8 C.
Samples should be analyzed shortly after the cells were prepared.
Special Note:
Indo-1 requires UV excitation. Make sure that you have an instrument with either an argon laser tuned to UV or a helium-cadmium laser available. Because experiments involving the measurement of calcium flux are performed directly on the flow cytometer, pre experiment consultation is strongly recommended.
June CH, Rabinovitch PS. Intracellular ionized calcium. Methods Cell Biol. 1994; 41 :149-74.
June CH, Abe R, Rabinovitch PS. Measurement of intracellular calcium ions by flow cytometry. In: Current Protocols in Cytometry, Vol 2, Robinson JP, Darzynkiewicz Z, Hyun W, Orfao A, Rabinovitch P, eds., John Wiley & Sons, 1997, pp. 9.8.1 – 9.8.19.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 29 Jan, 2007
You are reading this latest protocol version
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