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HEK293 Calcium Flux Assay

I. Introduction

This application note describes assay conditions for screening GPCR agonists on a LabChip 3000 Drug Discovery system using a HEK293 cell line.The miniaturized platform results in low cell and reagent consumption, high data quality and a high degree of automation. The LabChip microfluidic assay accesses test compounds from a microtiter plate and mixes them with the suspended cells continuously flowing through the chip. Flow through the microchannel network is generated by applying a vacuum to the waste wells of the chip. The system measures the fluorescence properties of individual cells flowing past a fluorescence detector.

Assay Summary

Total cell usage as low as 900 cells/well
Z' = 0.61 for agonist assay
Unattended run time of > 4 hours
Throughput of up to 2,500 samples in 4 hours

II. Experimental

Cell Culture

HEK293 cells from ATCC (CRL-1573) were grown in DMEM medium with 0.1 mM NEAA, 1mM sodium pyruvate, 10% Fetal Bovine Serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 2 mM L-glutamine.

Cell monolayers were grown in T150 flasks and were maintained at 5% CO2 and 37 oC and passaged at 50-70% confluency. To split cells, wash with PBS without Ca2+ or Mg2+. Aspirate PBS and add 2 mL of prewarmed Trypsin/EDTA and let stand for 30 s. Bang flask with hand to displace cells and verify cell lifting under microscope. Stop the Trypsin reaction by adding 10 mL of cell growth media and triturate cells. Seed a T150 flask at 2 x 106 cells/flask, diluting cells in prewarmed growth medium to a total volume of 24 mL. After 96 hours the flask will be 50-80% confluent. The day before use, seed a T75 flask with 5 x 106 cells, diluting cells in prewarmed growth medium to a total volume of 12 mL. Place two
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