Custom antibody project - tracking sheet reference #sa-2034 date: 2009-04-14

Accelerating Scientific Discovery
EliteTM Non-Wash Calcium Dye Assay

Calcium is essential for living organisms, in particular in cell physiology, where movement of the calcium ion Ca2+ into and out of the cytoplasm functions as a signal for many cellular processes. Calcium is the fifth-most-abundant element by mass in the human body, where it is a common cellular ionic messenger with many functions, and serves also as a structural element in bone. Calcium plays an important role in mediating the constriction and relaxation of blood vessels, nerve impulse transmission, muscle contraction, and hormone secretion. The serum level of calcium is closely regulated within a fairly limited range (9 to 10.5 mg/dL) in the human body. Both hypocalcemia and hypercalcemia are serious medical disorders. Causes of low calcium levels include chronic kidney failure, vitamin D deficiency, and low blood magnesium levels. Elite™ Non-Wash Calcium Assay Kit provides a simple method for detecting calcium in physiology solutions by using our proprietary red fluorescence probe. The fluorescence signal can be easily read by a fluorescence microplate reader at Ex/Em = 540/590 nm. The kit allows homogeneous measurement of intracellular calcium changes caused by activation of G-protein coupled receptors or calcium channels. The assay involves only one step of dye addition and does not require any washing steps. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to high throughput automation.

Continuous: Easily adapted to automation without a separation step.
Convenient: Formulated to have minimal hands-on time. No interference with magnesium.
Non-radioactive: No special requirement for waste disposal.
Kit Components
Component A: EliteTM Calcium Dye (light sensitive)
Component B: 10x Calcium Dye Signal Enhancer
Keep in freezer (-20 oC) and avoid exposure to light.
Materials Required (but not supplied)
 DMSO (Cat# D4540, Sigma) and Probenecid (Cat# P8761, Sigma).  96 or 384-well microplates: Tissue culture microplate with black wall and clear bottom is recommended.  A fluorescence microplate reader: Capable of monitoring fluorescence intensity at Ex/Em = 540±10/590±10 nm.
Assay Protocol for 96-Well/or 384-Well Plate

Thaw all the kit components to room temperature before starting the experiment.
1. Prepare the cell culture plate:
1.1 Seed 80 µl of cell suspension into each well of a 96-well plate or 20 µl of cell suspension into each well of a 1.2 Grow the cells overnight in a CO2 incubator  Please consider the environment before printing. Page 1
Tel: 1-800-919-0755  Fax: 1-240-683-5852  [email protected] Accelerating Scientific Discovery
2. Prepare assay buffers:
2.1 Prepare Buffer A (1X HBSS with 20 mM HEPES): 10 ml of 1M HEPES, pH 7.3 + 490 ml of 1X HBSS. Dissolve 142 mg of Probenecid in 1 ml of 1N NaOH. Add 160 µl of DMSO into the vial (Component A) containing 1 mg of calcium dye.
2.4 Prepare 2X Dye Loading Buffer (10 plates).
Add 8 ml of 10X Calcium Dye Signal Enhancer (Component B) into 72 ml of Buffer A.
Add 80 l of calcium dye stock solution. Mix well by vortexing. 3. Assay procesure:
3.1 Take the cell plate out from the incubator. 3.2 Add same volume of 2X Dye Loading Buffer into each well, 80 µl to a 96-well plate or 20 µl to a 384-well
3.3 Incubate at 37 C incubator for 1 hr. 3.4 Take the cells out of the incubator and leave at room temp (in the dark) for 30 min. 3.5 Put the plate into the instrument for assay For assays performed on a FlexStation (MDS), use the following wavelength parameters. Excitation: 485 nm; Emission: 530 nm; Cutoff 515 nm Note. Dispense speed and height for compound additions need to be optimized for each instrument.
Figure 1. Response of endogenous P2Y receptors to ATP. HEK293 cells were plated overnight in 20 μl culture medium on a 384
well Biocoat poly-D lysine coated plate. The next day, the cells were dye-loaded by adding 20 μl of 2X Dye Loading Buffer and
incubating for 1 hour at 37°C. ATP solution was added (10 μl/well) by a FLIPR Tetra (Molecular Devices), and the data was recorded
simultaneously. A. Kinetic curve of calcium response to different concentrations of ATP. B. ATP dose response curve (n = 4). EC50 =
1.1 μM.
Please consider the environment before printing. Page 2
Tel: 1-800-919-0755  Fax: 1-240-683-5852  [email protected]



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