Analysis of Calcium Flux by Flow Cytometry (CAT#: STEM-CBT-0067-WXH)

Introduction

Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity. Accurate measurements of intracellular Ca2+ concentrations allows for a more comprehensive understanding of Ca2+ regulated cell functions and signaling pathways. Ca2+ is an important molecule that affects a wide range of physiological and pathological processes. Unlike other secondary messengers, Ca2+ is not synthesized or metabolized, but stored and rapidly released by channels/pumps that maintain Ca2+ concentrations in distinct cellular compartments. Flow cytometry allows real time measurement of a calcium flux response utilizing fluorescent Ca2+ indicators which exhibit an increase in fluorescence upon binding to Ca2+.

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Principle Applications Procedure Materials Notes Related Products

Principle

The way this assay works basically involves a fluorochrome which undergoes some sort of structural confirmation change upon binding Calcium, resulting in either an increase/decrease in fluorescence or a change in fluorescence absorption/emission. So, if you load that dye into a cell sample, you can track this change in fluorescence over time and quantify the rate of flux (how fast calcium rushes in), peak calcium flux (how much calcium rushed in), or duration of flux (how long the flux lasted).

Applications

• Study of Ca2+ regulated cell functions and signaling pathways.
• Study the mechanisms of a range of physiological processes ( including the pathogenesis of diseases such as cancers and neurodegenerative disorders ).
• Determining the mechanism of action (MOA) of drugs prior to clinical trials.
• Assist in predicting potential unwanted drug side effects.
• Study of activation state of ion channels.
• Following the cell damage and apoptosis pathways.

Procedure

1.Prepare cell suspension
2.Add dye solution and incubate
3.Wash cells
4.Examine by flow cytometry

Materials

1.Ca2+ probes that increase fluorescence in the presence of free calcium: Fluo-3, Fluo-4
2.Ratiometric dyes: Indo-1, Fura-Red

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