Simultaneous Fluorescent Recordings of Extracellular ATP and IntracellularCalcium in Mammalian Cells.

Nicholas Mikolajewicz,Svetlana Komarova

doi:10.21769/bioprotoc.3242

Nicholas Mikolajewicz, Svetlana Komarova

Open Access

https://doi.org/10.21769/bioprotoc.3242

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Abstract

Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous monitoring can offer additional insights into P2 receptor physiology. This protocol takes advantage of the overlapping fluorescence spectra between the ATP-detection substrate luciferin and calcium indicator dye Fura2. Mammalian cells are loaded with Fura2-AM and live-cell recordings are acquired in the presence of a luciferin-luciferase imaging solution. This protocol allows to study stimulus-induced ATP release and directly relate changes in extracellular ATP concentration to observed calcium responses.

Highlights

[Background] ATP is a potent extracellular signaling molecule that is released in response to a variety of stress-related stimuli, including mechanical stimulation or injury (Mikolajewicz et al, 2018a)
Extracellular ATP is an autocrine and paracrine signal that acts through a number of purinergic (P2) receptors, which consist of two sub-families, P2X and P2Y receptors, and are omnipresent in virtually all mammalian cells (Burnstock and Verkhratsky, 2009)
This method is compatible with standard fluorescence microscopy and relies on the detection of a decrease in luciferin fluorescence emission intensity when it is depleted in the presence of ATP (Table 1)

Summary

Introduction

[Background] ATP is a potent extracellular signaling molecule that is released in response to a variety of stress-related stimuli, including mechanical stimulation or injury (Mikolajewicz et al, 2018a). Since the excitation and emission spectra of luciferin overlap with Fura[2] (Figure 1), and the dyes are compartmentalized in the extracellular (luciferin) and intracellular (Fura2) spaces, this overlap in fluorescence spectra can be used to simultaneously measure extracellular ATP concentrations and changes in intracellular [Ca2+]i, as reported in our prior work (Mikolajewicz et al, 2018b) and presented in detail below. Fura2-AM, cell permeant (Thermo Fisher Scientific, Invitrogen, catalog number: F1221) 8.

Results

Conclusion