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Cell Meter™ Flow Cytometric Calcium Assay Kit

The ATP induced intracellular calcium release was measured by Cell Meter™ Flow Cytometric Calcium Assay Kit in CHO-K1 cells. Cells were incubated with Calbryte™ 520 AM dye for 30 min at 37 °C before 10 µM ATP was added into the cells. The baseline was acquired and the rest of the cells were analyzed after the addition of ATP. The response was measured over time. The analysis was done on NovoCyte™ 3000 Flow Cytometer. The arrows on the graph indicate the time (30 sec) between addition of ATP and the actual analysis.
The ATP induced intracellular calcium release was measured by Cell Meter™ Flow Cytometric Calcium Assay Kit in CHO-K1 cells. Cells were incubated with Calbryte™ 520 AM dye for 30 min at 37 °C before 10 µM ATP was added into the cells. The baseline was acquired and the rest of the cells were analyzed after the addition of ATP. The response was measured over time. The analysis was done on NovoCyte™ 3000 Flow Cytometer. The arrows on the graph indicate the time (30 sec) between addition of ATP and the actual analysis.
The ATP dose dependent intracellular calcium release was measured by Cell Meter™ Flow Cytometric Calcium Assay Kit in CHO-K1 cells. Cells were incubated with Calbryte™ 520 AM dye for 30 min at 37 °C before ATP was added into the cells. The baseline was acquired and the rest of the cells were analyzed after the addition of ATP. The response was measured over time. The analysis was done on NovoCyte™ 3000 Flow Cytometer. <strong>A</strong>. 10 µM, 1 µM or 0 µM ATP were added to the cells. The arrows on the graph indicate the time (30 sec) between addition of ATP and the actual analysis. <strong>B</strong>. Time-dependent changes of fluorescent signal.
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Catalog Number36310
Unit Size
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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
Excitation (nm)493
Emission (nm)515
Quantum yield0.751
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
493
Emission (nm)
515
Quantum yield
0.751
Cell Meter™ Flow Cytometric Calcium Assay Kit provides fluorescence-based assays for detecting intracellular calcium mobilization using a flow cytometer. It can be used for kinetic reading or for endpoint reading. After loading the Calbryte™ 520 AM dye into cells of interest, simply wash the cells and add the calcium flux agonist, one can then read the sample via a flow cytometer using kinetic reading mode or endpoint reading mode. Calbryte™ 520 AM can cross cell membrane passively by diffusion. Once inside the cells, the lipophilic blocking groups of Calbryte™ 520 AM are cleaved by esterase, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced 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 Calbryte™ 520. The Cell Meter™ Flow Cytometric Calcium Assay Kit can be used for monitoring cellular calcium flux as well as cell sorting.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Other instruments

ArrayScan, FDSS, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux

Components


Component A: Calbryte™ 520 AM1 vial, lyophilized
Component B: Assay Buffer1 bottle (50 mL)
Component C: Probenecid 1 bottle (10 mL, 25mM)
Component D: HHBS 1 bottle (100 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in Assay Buffer
  2. Add Calbryte™ 520 AM dye-loading solution (1 µL)
  3. Incubate at 37°C for 30 minutes
  4. Wash the cells
  5. Add calcium flux stimulator
  6. Monitor fluorescence intensity with flow cytometer using 530/30 nm filter (FITC channel)

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.

Calbryte™ 520 AM stock solution (500X):
Add 100 µL of DMSO (Not provided) into the vial of Calbryte™ 520 AM stock solution (Component A) and mix them well. Note: 100 µL of Calbryte™ 520 AM stock solution is enough for 100 assays. Unused Calbryte™ 520 AM stock solution can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Remove cell culture medium and add 0.5 mL of Assay Buffer. Note: For adherent cells and non-adherent cells, 4 X105 – 8 X105 and 1X10- 2X106 are recommended to use, respectively. Each cell line should be evaluated on the individual basis to determine the optimal cell density for the intracellular calcium mobilization.

  2. Add 1 µL Calbryte™ 520 AM stock solution (500X) into 0.5 mL non-adherent or adherent cells in Assay Buffer (Component B). Note: If your cells (such as CHO cells) contain organic anion-transports, then probenecid (Component C) may be added to the dye working solution (final in well concentration will be 0.125-1 mM) to reduce leakage of the de-esterified indicators.

  3. Incubate the cells at 37°C for 30 minutes.

  4. For non-adherent cells, centrifuge the cells and remove the dye. Re-suspend the cells in 0.4 mL HHBS (Component D). For adherent cells, use 0.5 mM EDTA to gently lift the cells from the plate and centrifuge. Re-suspend the cells in 0.4 mL HHBS (Component D). Note: For detaching adherent cells from the plate, enzymatic reagents (e.g. trypsin, Accutase) can be considered but need to be tested to make sure the receptor of interest on the cell surface is not affected.

  5. Prepare 5X agonist compound with HHBS or your desired buffer.

  6. Analyze the sample before and after the addition of 100 µL of the prepared agonist on a flow cytometer using 530/30 nm filter (FITC channel). 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

Excitation (nm)493
Emission (nm)515
Quantum yield0.751

Citations


View all 1 citations: Citation Explorer
Inhibition of P110$\alpha$ and P110$\delta$ catalytic subunits of PI3 kinase reverses impaired arterial healing after injury in hypercholesterolemic male mice
Authors: Chaudhuri, Pinaki and Smith, Andrew H and Graham, Linda M and Rosenbaum, Michael A
Journal: American Journal of Physiology-Cell Physiology (2021): C943--C955

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