Miniaturization of a Calcium Mobilization Assay in 384 Well Format

Life
Sciences
Miniaturization of a Calcium Mobilization
Assay in 384 Well Format
SnAPPShots
A brief report
from the Corning
Applications Group
Yu Alice Gao and Debra Hoover
Introduction
Calcium mobilization assays are frequently used to investigate G-protein couple receptor
(GPCR) and ligand interactions and to screen library compounds or drug candidates that
interact with GPCR targets. Currently, these assays are predominantly performed in a
384 well format with total volumes ranging from 50 to 100 µL.
In this study, we show a calcium mobilization assay that has been miniaturized to 25 to
40 µL using a new 384 well low volume (LV) black clear bottom (BCB) microplate from
Corning.The results demonstrate that the quality of the data and assay performance on
this LV microplate are comparable to that obtained from 384 well normal volume (NV)
microplates.
Material and Methods
Transfected CHO-K1 cells (M1WT2 from ATCC, CRL-1984) were used in this study.
This cell line over expresses rat M1 muscarinic acetylcholine receptor at the level of
~370 fmole of receptor protein per mg of membrane protein.
Corning ® 384 well LV BCB microplates (Cat. No. 3542) allow for assay volumes ranging
from 5 to 50 µL. The microplates have a well bottom area of 0.032 cm 2 , roughly half that
of the NV 384 well microplates, and are FLIPR ® (Molecular Devices, Inc.) compatible.

The LV microplates were seeded at 5,000 cells per well in 10 µL medium (HAMs F-12
with 10% FBS and 50 µg/mL g418). All cultures were grown overnight in a humidity
controlled incubator with 5% CO 2 at 37°C. A calcium mobilization assay was then performed
with these cultures using a Calcium 3 kit (Molecular Devices, Inc.). The procedure
was as follows: After the addition of 10 µL calcium dye solution to wells containing cells,
the microplates were incubated for 30 min in a humidity controlled incubator with 5%
CO 2 at 37°C. The microplates were then equilibrated to room temperature for 30 min
before being loaded into a Flexstation ® reader (Molecular Devices, Inc.). The fluidic
module on the FlexStation was programmed to transfer 5 µL Carbachol solution to
induce the calcium response or 5 µL of plain buffer for negative controls. Final volume
of each well was 25 µL. On NV microplates, cell seeding density was doubled (10,000
cells per well in 20 µL medium). Volumes were 20 µL for calcium dye and 10 µL for
Carbachol. Final volume was 50 µL per well.
The calcium signal was monitored over a period of 1 min at Excitation and Emission
wavelengths of 485 and 525 nm.
Results
The performance of LV microplates was compared to NV microplates in three metrics.
First was the time-dependent calcium flux kinetics upon the addition of Carbachol solution.
As shown in Figure 1, the calcium flux responses were very similar between cells
grown on LV and NV microplates. These responses were rapid and consistent over all
the experiments conducted. Because of reduced cell numbers and reagent volumes, the
overall signal strength on the LV microplate was reduced, with a signal to background
ratio of 18 to 20. The signal to background ratio from NV microplates was 25 to 30.
Nevertheless, the assay window from the LV microplates was still statistically sound and
such reduction did not affect overall performance of the assay as demonstrated below.
The second metric compared in this study was the dose response curve to the agonist
Carbachol. As shown in Figure 2, the dose response curve and estimated EC 50 to
carbachol stimulation were very similar between LV and NV microplates.
60000
50000
+ Carbachol (10 µM)
or buffer
120000
100000
+ Carbachol (10 µM)
or buffer
40000
80000
RFU
30000
RFU
60000
40000
20000
LV microplate
20000
NV microplate
10000
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Time (sec.)
Time (sec.)
Figure 1. Comparison of time-dependent calcium mobilization upon the addition of Carbachol. The kinetic
response obtained from the LV plate is very similar to that from NV plate, although the overall signal
strength and background is reduced on the LV plate.

40000
80000
LV microplates
NV microplates
30000
60000
Max – Min (RFU)
20000
Max – Min (RFU)
40000
10000
20000
EC 50 = 100 nM
EC 50 = 93 nM
0
0
-9.5 -8.5 -7.5 -6.5 -5.5 -4.5 -9.5 -8.5 -7.5 -6.5 -5.5 -4.5
Log [Carbachol]
Log [Carbachol]
Figure 2. Comparison of the dose response curve between the LV and the NV microplates. The estimated
EC 50 from the LV microplate for Carbachol was equivalent to that obtained from the NV microplate.
Each data point was the average of five replicates.
Finally, an analysis of the across-plate performance was examined and is presented in
Table 1. The results showed that the across-plate variation for the LV microplates was
smaller compared to that for the NV microplates, resulting in an enhanced overall assay
performance on the LV microplates (expressed as Z').
Table 1. Overall performance comparison of the calcium mobilization assay on the
LV and the NV microplate
Maximal Signal (RFU) Background (RFU) Z'
LV plate 26040 (+3647) 1335 (+389) 0.51
NV plate 46783 (+7986) 1778 (+415) 0.44
Numbers are the average and standard deviation from 96 replicates (one quadrant).
Conclusions
◗ Corning ® 384 well low volume black clear bottom microplates (Cat. No. 3542) perform
well in a calcium mobilization assay. All three performance metrics (calcium response
kinetics, pharmacological profile of a known agonist for the receptor, and Z') for the
LV microplates are comparable to normal volume microplates.
◗ The 384 well low volume black clear bottom microplates offer significant reagent
savings (50%) as well as reduced cell preparation work and are ideal for miniaturization
of cell based assays in the 384 well format.

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