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HEK293 Calcium Flux Assay

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
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Source:


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