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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 T75 flasks per unattended run in an incubator at 10% CO2 in air and 37 oC. The flask will be 50-80% confluent the next day and ready for use.

Cell Labeling and Suspension

Label cells with Fluo-4 and Fura Red by washing gently with 5 mL PBS without Ca2+ or Mg2+ in a T75 flask. Add 1.5 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 5 mL of cell growth media. Triturate cells and add to a 15 mL tube. Triturate again with pipette several times. Perform cell count, Centrifuge cells at 1400 rpm for 2 min. Aspirate supernatant and flick cell pellet to loosen cells. Add 1 mL of dye loading solution (see Section V) and triturate vigorously to break cell clumps. Add remainder of dye loading solution to the cells for a final cell density of 1.5 x 106 cells/mL and again triturate vigorously. Place the cells in a T75 flask and place in an incubator at 5% CO2 and 37 oC for 45 min.

Transfer cells to a 15mL tube and centrifuge at 1400 rpm for 2 min. Aspirate the supernatant and loosen the cell pellet by flicking. Add 1 mL of cell wash buffer (see Section V) with pipette and triturate vigorously to break cell clumps. Add 6 mL of cell wash buffer and triturate vigorously again. Repeat aspiration, centrifugation and washing once more. Centrifuge sample at 1400 rpm for 2 min. Aspirate supernatant and loosen pellet by flicking. Resuspend cell pellet in 1 mL of storage buffer (see Section V) and triturate vigorously to break cell clumps. Add 4 mL of cell storage buffer and triturate vigorously again. Count cells and centrifuge at 1400 rpm for 2 min. Aspirate supernatant and loosen pellet by flicking. Add 1 mL of cell assay buffer (see section V) with pipette and triturate vigorously to break cell clumps Add remainder of cell assa y buffer to the cells for a final cell density of 2.3 x 106 cells per mL and again triturate vigorously.

III. Assay Development

Kinetic Response

The LabChip 3000 system is designed such that calcium response for each cell is measured at a fixed incubation time. The cell velocity and the length of the incubation channel determine the incubation time. Cell velocity is a function of the pressure applied to the chip, the chip temperature and the solution viscosity; while incubation channel length can be varied by selecting detection points at three locations (or zones) along the cell path. Typically pressures of -1.2, -2 and -3 psi are sufficient to gather statistics on optimum incubation time. In this experiment, carbachol was sipped up onto the chip and contacted with cells. The ratio of fluorescence for each cell at 530 nm (Fluo-4) and 685 nm (Fura Red) was measured and plotted as a function of incubation time. From Figure 1, 30 s was determined to be the optimum incubation time for HEK293 cells stimulated by carbachol. Table 1 gives the assay conditions for the HEK293 agonist assay.

Assay Stability

Once reaction conditions were determined, the stability of the assay over the anticipated run time was assessed. Stability of the assay was assessed by examining the cell count rate and the cell viability (e.g. EC50) over time. Figure 2 shows that cell counts are > 80 cells per well after 4 hr. Figure 3 shows the stability of the cell response over a typical run time of 4 hr.

IV. Assay Validation

Typical validation criteria include data quality (Z') assessment, assay stability over multiple runs, and cell usage.

Data Quality Assessment

Figure 4 shows the calculated Z' values for the agonist assay. Ass ay stability was measured over two runs and was determined to be adequate over the intended run time of 4 hr. Average Z' value was to 0.61 over 4 hr using plates with carbachol agonist controls.

Assay Stability

In addition to Z' analysis, the stability of the agonist response was measured over several runs. Both % activity and EC50 demonstrate assay stability over time and from run to run.

V. Assay Conditions

The following buffers were used for screening:

BASE BUFFER
10 mM HEPES, pH 7.4
130 mM NaCl
5 mM KCl
10 mM Glucose

WASH BUFFER* 1X Base Buffer 0.1% Pluronic F-68 1 mM Probenecid

CELL STORAGE BUFFER*
1X Base Buffer
2 mM CaCl2
1 mM MgCl2
1 mM Probenecid

PLATE/TROUGH BUFFER*
1X Base Buffer
40 mM NaCl 2 mM
CaCl2 1 mM
MgCl2 0.1%
Pluronic F-68

DYE LOADING SOLUTION*
a) Add 15 μL of 1 mM Fura Red AM to 15 μL of Pluronic F127
b) Add 15 μL of 1 mM Fluo-4 AM to 15 μL of Pluronic F127
c) Combine a) with 5 mL DMEM
d) Combine b) with c)
e) Add 10 μL of 0.5 M Probencecid

* Make fresh daily

ROW MARKER (IN TROUGH BUFFER)*
0.25 μM Fluorescein

100% CONTROL ON PLATE ( AGONIST)*
200 μM Carbachol

Load cells with densities of 2.25 x 106 cells/mL with 200-250 μL per well.
Operate the system a t -2 psi at detection zone 2 and collect data at 100 Hz.
Use the LabChip 3000 system with blue laser excitation and green/red fluorescence detection (CCD2, CCD3).

A job file, which includes all LabChip 3000 Drug Discovery system operating parameters for this assay, can be downloaded at www.caliperLS.com


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