Time-Dependent Measurement of Intracellular Calcium Levels in Live Cells by Flow Cytometry

December 02, 2015

George Washington University, Washington DC
Surgery
Post-doctoral Fellow

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Company:

Thermo Fisher Scientific

Product Name:

Fluo-3, AM, Calcium Indicator

Catalog Number:

F-1242

Intracellular calcium levels are key indicators of any signaling system involving Ca2+ as second messenger. Stimulation of cells is involved with calcium influx across the cell membrane through ionophores and native calcium channels. This calcium influx or altered intracellular calcium levels can be measured using a dye, Fluo-3, which becomes fluorescent upon binding to calcium. The AM is acetoxymethyl ester which facilitates the transport of the dye across the membrane and once inside the cell, is broken down by non-specific esterase to free the Flou-3 dye. Detection of intracellular Ca2+ is useful in comparing time-dependent dynamics of intracellular signal transduction.

Experimental Design and Results Summary

Application

Flow cytometry; fluorescence microscopy

Starting Material

Live cells

Protocol Overview

Fluo-3 AM dye was dissolved in DMSO to get a stock of 5mM concentration. Just prior to application it was diluted (1:1000) in buffer (PBS) or appropriate media to a conc. of 5uM.The cells to be analyzed for intracellular calcium (different groups) were washed in staining buffer for FACS. We used cells from mouse splenocytes. We wanted to compare different level of Ca2+ influx in T cells cultured with various cytokines. Thus, we decided to activate cells with non-specific PMA/ionomycin to measure Ca2+ influx. Initially cells (1X10e6) were resuspended in FACS buffer and acquisition was done for about 30-40sec. for the cells which were unstimulated (basal level). Without turning off the acquisition process, the tube was taken out and PMA/ionomycin was added (stimulation), and immediately the tube was placed back for acquisition. We saw a rise in the Fluo-3 index, indicating an influx of intracellular Ca2+ and increased levels in the cytosol. The amount of florescence is directly proportional to the Ca2+ concentration and thus can be compared across groups.

Tips

Makes at least a 1000-fold dilution of the stock as it is dissolved in DMSO.

Results Summary

Increased levels of Ca2+ was observed after stimulation as indicated by the increase in Fluo-3 fluorescence.

Additional Notes

Stimulation should be done as fast as possible after taking out the tube from acquisition and put back immediately for continual analysis. As this is a dynamic evaluation, any delay will alter results.

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Summary

The Good

Unique method of intracellular Ca2+ estimation by flow cytometry

The Bad

The process needs to maintain equal time lapses to compare results.

The Bottom Line

Unique method, which is achievable under normal laboratory set-up, to measure intracellular Ca2+ which can be an immensely important second messenger in cell signaling pathway.

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