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No Wash Calcium Assay Product Specifications

ION Biosciences Brilliant Calcium Assay is a total assay solution for multi-well plate-based, high-throughput measurements of dynamic changes in intracellular Ca2+. The ION Biosciences Brilliant Calcium Assay is useful for a wide range of effectors of intracellular calcium including many G protein-coupled receptors (GPCRs), divalent cation channels, and other proteins capable of transporting Ca2+ or affecting intracellular Ca2+ dynamics. In multi-well, plate-based formats, the Brilliant Calcium Assay can be used to discover and characterize the effects of tens to many tens-of-thousands of compounds and environmental factors which can act to affect intracellular Ca2+ levels. Over the past 20 years, fluorescence-based measures of intracellular Ca2+ have been widely utilized to discovery small-molecule modulators of a host of GPRCs, Ion channels and other targets of interest for both drug discovery and basic research. Inside the ION Brilliant Assay package, you will find all the reagents necessary for preparation of the Brilliant Calcium Assay for use as a washed or no wash assay for adherent and non- adherent cells. The addition of a Probenecid solution and an extracellular background masking solution (TRS) offers users the ultimate in compatibility for cells types which are difficult to load with fluorescent Ca2+ indicators (e.g. Chinese Hamster Ovary, CHO cells) and when performing assays in complete, serum- containing, cell-culture medium is desired.  
Figure 1. HEK-293 cells natively expressing the M3 muscarinic acetylcholine receptor, loaded with ION Brilliant Calcium Assay reagent before (A) and after (B) exposure to 30 μM acetylcholine.

Figure 1. HEK-293 cells natively expressing the M3 muscarinic acetylcholine receptor, loaded with ION Brilliant Calcium Assay reagent before (A) and after (B) exposure to 30 μM acetylcholine.

 
Figure 2. Acetylcholine-evoked increases in intracellular Ca2+ in HEK-293 cells natively expressing M3 muscarinic acetylcholine receptor. ION Brilliant Calcium No Wash Assay and a leading no wash calcium kit were evaluated according to manufacturer’s instructions. ______ 30 μM acetylcholine ------ 0.1 μM acetylcholine

Figure 2. Acetylcholine-evoked increases in intracellular Ca2+ in HEK-293 cells natively expressing M3 muscarinic acetylcholine receptor. ION Brilliant Calcium No Wash Assay and a leading no wash calcium kit were evaluated according to manufacturer’s instructions.
______ 30 μM acetylcholine ------ 0.1 μM acetylcholine

Figure 3. Concentration-dependent increases in intracellular Ca2+ in HEK-293 cells natively expressing the M3 muscarinic acetylcholine receptor. ION Brilliant Calcium No Wash Assay and a leading no wash calcium kit were evaluated according to manufacturer’s instructions.

Figure 3. Concentration-dependent increases in intracellular Ca2+ in HEK-293 cells natively expressing the M3 muscarinic acetylcholine receptor. ION Brilliant Calcium No Wash Assay and a leading no wash calcium kit were evaluated according to manufacturer’s instructions.

 
Package Contents
Description
Storage Temperature

Reagent A

Brilliant Calcium Indicator

-20°C

Reagent B

DySolv

room temperature

Reagent C

Assay Buffer

4°C

Reagent D

Probenecid Solution

4°C

Reagent E

TRS Solution

4°C

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No Wash Calcium Assay Single Microplate Kit Basic Protocol (Adherent Cells)

  1. Prepare Brilliant Calcium Indicator Solution
    1. Add 50 μL DySolv (Reagent B) to the tube containing Brilliant Calcium Indicator (Reagent A)
    2. Vortex until Reagent A is fully dissolved
    3. Transfer the Reagent A solution to the bottle containing Assay Buffer (Reagent C) and vortex briefly
    4. OPTIONAL1 Add 100 μL Probenecid (Reagent D) to the bottle containing the Reagent C solution and vortex briefly
    5. OPTIONAL2 Add 400 μL TRS (Reagent E) to the bottle containing the Reagent C solution and vortex briefly
  2. Assay
    1. Remove cell culture medium from cell-containing plate
    2. Add Brilliant Calcium Indicator Solution from step 1 (80 μL/well for 96-well plates, 20 μL/well for 384-well plates)Protect from direct light. Incubate for 1 hour at room temperature.
    3. Collect data3 using an appropriate fluorescent plate reader with liquid handling capabilities (WaveFront Panoptic, Hamamatsu FDSS, Molecular Devices FLIPR, Molecular Devices Flexstation) equipped with filters designed for fluorescein-type fluors (excitation ~490/emission ~520 nm)

1 The use of Probenecid is indicated for some cell types to counteract these cells’ propensity to extrude Brilliant Calcium and other similar indicator dyes resulting in poor dye retention.

2 The TRS reagent is useful for suppressing background resulting from fluorescent compounds in the solutions surrounding the cells. Use of TRS may result in improved signal-to-noise.

3 Data collection typically includes the addition of test compounds or additional reagents to the assay plate during the course of data acquisition. Volumes and concentrations of test compounds and additional reagents vary. Commonly used addition volumes are 5, 10, 20 μL for 384-well plates and 20, 40, and 80 μL/well for 96-well plates.

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No Wash Calcium Assay Ten Microplate Kit Basic Protocol (Adherent Cells)

  1. Prepare Brilliant Calcium Indicator Solution
    1. Add 500 μL DySolv (Reagent B) to the tube containing Brilliant Calcium Indicator (Reagent A)
    2. Vortex until Reagent A is fully dissolved
    3. Transfer the Reagent A solution to the bottle containing Assay Buffer (Reagent C) and vortex briefly
    4. OPTIONAL1 Add 1000 μL Probenecid (Reagent D) to the bottle containing the Reagent C solution and vortex briefly
    5. OPTIONAL2 Add 4 mL TRS (Reagent E) to the bottle containing the Reagent C solution and vortex briefly
  2. Assay
    1. Remove cell culture medium from cell-containing plate
    2. Add Brilliant Calcium Indicator Solution from step 1 (80 μL/well for 96-well plates, 20 μL/well for 384-well plates)
    3. Protect from direct light. Incubate for 1 hour at room temperature.
    4. Collect data3 using an appropriate fluorescent plate reader with liquid handling capabilities (WaveFront Panoptic, Hamamatsu FDSS, Molecular Devices FLIPR, Molecular Devices Flexstation) equipped with filters designed for fluorescein-type fluors (excitation ~490/emission ~520 nm)

1 The use of Probenecid is indicated for some cell types to counteract these cells’ propensity to extrude Brilliant Calcium and other similar indicator dyes resulting in poor dye retention.

2 The TRS reagent is useful for suppressing background resulting from fluorescent compounds in the solutions surrounding the cells. Use of TRS may result in improved signal-to-noise.

3 Data collection typically includes the addition of test compounds or additional reagents to the assay plate during the course of data acquisition. Volumes and concentrations of test compounds and additional reagents vary. Commonly used addition volumes are 5, 10, 20 μL for 384-well plates and 20, 40, and 80 μL/well for 96-well plates.

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