Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit *Optimized for microplate reader*

The ATP dose dependent intracellular calcium release was measured by Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit in CHO-K1 cells in a 96-well plate. 3 columns of cells were incubated with Fluo-8E™ AM dye loading solution for 1 hr at 37°C before ATP was added into all 3 columns of the wells. The plate was read immediately after the addition of the ATP by ClarioStar (BMG Labtech) at Ex/Em = 490/525 nm with bottom and endpoint read mode.
The ATP dose dependent intracellular calcium release was measured by Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit in CHO-K1 cells in a 96-well plate. 3 columns of cells were incubated with Fluo-8E™ AM dye loading solution for 1 hr at 37°C before ATP was added into all 3 columns of the wells. The plate was read immediately after the addition of the ATP by ClarioStar (BMG Labtech) at Ex/Em = 490/525 nm with bottom and endpoint read mode.
Ordering information
Price ()
Catalog Number36312
Unit Size
Find Distributor
Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (260 nm)1.076
Correction Factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)23430
Excitation (nm)495
Emission (nm)516
Quantum yield0.161
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
1.076
Correction Factor (280 nm)
0.769
Extinction coefficient (cm -1 M -1)
23430
Excitation (nm)
495
Emission (nm)
516
Quantum yield
0.161
Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit enables homogeneous fluorescence-based assays for detecting intracellular calcium mobilization without the need to use kinetics reading mode. It can be used on Fluorescence microplate readers with bottom read mode that do not have a build-in liquid dispenser or the ability of kinetics reading. After loading the Fluo-8E™AM dye into cells of interest, without wash steps, one can simply add the calcium flux agonist by a liquid dispenser or hand pipetting, and then read the plate by a fluorescent reader. Fluo-8E™ AM can cross cell membrane passively by diffusion. Once inside the cells, the lipophilic blocking groups of Fluo-8E™ AM are cleaved by esterase, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced and long lasting upon binding to calcium. When cells expressing GPCR of interest are stimulated with an agonist, the receptor signals the release of intracellular calcium, which significantly increases the fluorescence of Fluo-8E™. The characteristics of its high sensitivity, >100 times fluorescence enhancement and long lasting fluorescent signal make Fluo-8E™ an ideal indicator for the measurement of cellular calcium on fluorescence microplate readers that do not have the fluid transfer and kinetic reading mode capability. The Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit can be performed in a 96-well or 384-well microtiter-plate format.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateBlack wall/Clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: Fluo-8E™ AM2 vials, lyophilized
Component B: 10X Pluronic® F127 Plus1 bottle (1 mL)
Component C: HHBS (Hanks’ with 20 mM Hepes)1 bottle (20 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Add Fluo-8E™ AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at 37°C for 60 minutes
  4. Add 50 µL calcium flux stimulator
  5. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Fluo-8E™ AM stock solution:
Add 10 µL of DMSO into the vial of Fluo-8E™ AM (Component A) and mix well. Note: 10 µL of Fluo-8E™ AM stock solution is enough for 50 assays (Half of the 96-well plate).  Note: Unused Fluo-8E™ AM stock solution can be aliquoted and stored at < -20 oC for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

2. Assay Buffer (1X):
Add 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B), and mix them well. Note: 10 mL of Assay Buffer (1X) is enough for one plate. Aliquot and store unused 1X assay buffer at < -20 °C. Protect from light and avoid repeated freeze-thaw cycles. 

PREPARATION OF WORKING SOLUTION

Fluo-8E™ AM dye-loading solution:
Add 10 µL of Fluo-8E™ AM stock solution into 5 mL of Assay Buffer (1X), and mix them well. Note: This working solution is stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-8E™ AM dye-loading solution into the cell plate. Do not remove the growth medium from the cell plate.

  2. Incubate the dye-loading plate in a 5% CO2 incubator at 37oC for 45 - 60 minutes.
     
  3. Prepare the Calcium stimulator solution (5X) with HHBS or your desired buffer.

  4. Add 50 µL of the prepared stimulator and run the calcium flux assay immediately by monitoring the fluorescence intensity at Ex/Em = 490/525 nm (Cutoff=515 nm) with bottom read mode. Note: To achieve the best results, it is important to run the assay within 1 minute after the addition of the agonist. It is also important to make sure the time between the agonist addition and the beginning of the actual reading stays constant for all the samples.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)1.076
Correction Factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)23430
Excitation (nm)495
Emission (nm)516
Quantum yield0.161

Citations


View all 1 citations: Citation Explorer
Naloxone Is a Potential Binding Ligand and Activator of the Capsaicin Receptor TRPV1
Authors: Melkes, Barbora and Markova, Vendula and Hejnova, Lucie and Marek, Ales and Novotny, Jiri
Journal: Biological and Pharmaceutical Bulletin (2020): 908--912

References


View all 34 references: Citation Explorer
Fluorescence absorbance inner-filter decomposition: the role of emission shape on estimates of free Ca(2+) using Rhod-2
Authors: Territo PR, Heil J, Bose S, Evans FJ, Balaban RS.
Journal: Appl Spectrosc (2007): 138
Protein kinase C and myocardial calcium handling during ischemia and reperfusion: lessons learned using Rhod-2 spectrofluorometry
Authors: Stamm C, del Nido PJ.
Journal: Thorac Cardiovasc Surg (2004): 127
Novel fluo-4 analogs for fluorescent calcium measurements
Authors: Martin VV, Beierlein M, Morgan JL, Rothe A, Gee KR.
Journal: Cell Calcium (2004): 509
Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux
Authors: Bednar B, Cunningham ME, Kiss L, Cheng G, McCauley JA, Liverton NJ, Koblan KS.
Journal: J Neurosci Methods (2004): 247
Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry
Authors: Stamm C, Friehs I, Choi YH, Zurakowski D, McGowan FX, del Nido PJ.
Journal: Cardiovasc Res (2003): 695
Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Biophys J (2001): 549
Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Cell Calcium (2001): 217
Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions
Authors: Lannergren J, Westerblad H, Bruton JD.
Journal: J Muscle Res Cell Motil (2001): 265
Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy
Authors: MacGowan GA, Du C, Glonty V, Suhan JP, Koretsky AP, Farkas DL.
Journal: J Biomed Opt (2001): 23
Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death
Authors: Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC, Agid Y, Ruberg M.
Journal: J Comp Neurol (2000): 297