Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Summary

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Abstract

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca²⁺-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca²⁺ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca²⁺-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

Protocol

1. Cell preparations in 96-well plates

For HEK293 cells transiently trasfected with human TRPA1 cDNA

1. HEK293 cells are grown for one to two days in Dulbecco's Minimal Essential Medium (DMEM) containing 4.5 mg/ml glucose (Thermo Scientific, SH3002201), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, 16000-044), 50 units/ml penicillin and 50 μg/ml streptomycin (Invitrogen, 15140-163). On the day of the transfection, the cell density should reach 70-90%.
2. Coat 96-well plates by pipetting 50 μl poly-L-ornithine (Sigma P3655, 20 μg/ml in sterile distilled water) to each well and incubating at 37°....

Discussion

Intracellular Ca²⁺ plays important roles in many physiological and pathological conditions, such as neurotransmitter release (3), cardiac action potential (4), muscle cell contraction (5), cell signal transduction (6-7), and excitotoxic cell death (8). Measurement of intracellular Ca²⁺ levels helps to elucidate functional changes of Ca²⁺-permeable channels in the plasma membrane or intracellular organelles and the contribution of
Ca²⁺ to the above processes (9-13). The as.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FlexStation 3	Molecular Devices	FLEX3
96-Well, FlexStation Pipet Tips	Molecular Devices	9000-0912
96-Well Plates	Greiner Bio-One	655090
96-Well Clear Flat Bottom plates	Costar	3595
DMEM	Invitrogen	12430-104
Opti-MEM	Invitrogen	31985-070
Lipofectamine 2000	Invitrogen	11668-019
Fluo-4 AM	Invitrogen	F14202
PluronicF-127	Invitrogen	P3000MP
Poly-L-ornithine	Sigma-Aldrich	P36551
probenecid	Sigma-Aldrich	P8761
Bovine serum albumin	Sigma-Aldrich	A9418