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Cell Meter™ Cell Viability Assay Kit *Green Fluorescence*

CHO-K1 cell number response was measured with Cell Meter™ Cell Viability Assay Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 µL/well of dye-loading solution for 1 hour at 37°C. The fluorescence intensity was measured at Ex/Em = 490/ 525 nm with NOVOstar instrument (from BMG Labtech). The fluorescence intensity was linear (R square = 1) to the cell number as indicated.
CHO-K1 cell number response was measured with Cell Meter™ Cell Viability Assay Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 µL/well of dye-loading solution for 1 hour at 37°C. The fluorescence intensity was measured at Ex/Em = 490/ 525 nm with NOVOstar instrument (from BMG Labtech). The fluorescence intensity was linear (R square = 1) to the cell number as indicated.
Ordering information
Price ()
Catalog Number22786
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)494
Emission (nm)514
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
494
Emission (nm)
514
Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used for monitoring cell viability. This kit uses the non-fluorescent calcein AM that becomes strongly fluorescent upon entering into live cells. Calcein AM is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the non-fluorescent calcein AM by intracellular esterases generates the strongly fluorescent hydrophilic calcein that is well-retained in the cell cytoplasm. The esterase activity is proportional to the number of viable cells, and thus directly related to the fluorescence intensity of calcein generated from the esterase-catalyzed hydrolysis of calcein AM. Cells grown in black-walled plates can be stained and quantified in less than two hours. The assay is more robust than the tetrazolium salt or Alarmar Blue™-based assays. It can be readily adapted for high-throughput assays in a wide variety of fluorescence platforms such as microplate assays, immunocytochemistry and flow cytometry. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. Using 100 uL of reagents per well in a 96-well format, this kit provides sufficient reagents to perform 500 assays. Using 25 uL of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 2000 assays.

Platform


Fluorescence microplate reader

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

Components


Component A: CytoCalcein™ Green5 vials, lyophilized
Component B: DMSO1 vial (200 µL)
Component C: Assay Buffer1 bottle (50 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds
  2. Add the same volume of working solution as the cell medium
  3. Incubate at room temperature or 37°C for 1 hour 
  4. Monitor fluorescence with fluorescence microplate reader at Ex/Em= 490/525 nm (Cutoff=515 nm)

Important notes
Thaw one of each kit component at room temperature before use.

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. CytoCalcein™ Green stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Green (Component A) and mix well. Protect from light. Note: 20 µL of CytoCalcein™ Green stock solution is enough for one plate.

PREPARATION OF WORKING SOLUTION

Add the whole content (20 µL) of CytoCalcein™ Green stock solution into 10 mL of Assay Buffer (Component C) and mix well. Note: The working solution is stable for at least 2 hours at room temperature. Note: If the cells, such as CHO cells, contain organic-anion transporters which cause the leakage of the fluorescent dye over time, a probenecid stock solution should be prepared and added to the loading buffer at a final in-well working concentration of 1 - 2.5 mM.

SAMPLE EXPERIMENTAL PROTOCOL

Cells Preparation:

Plate 100 to 100, 000 cells/well in a tissue culture microplate with black wall and clear bottom. Add test compounds into the cells and incubate for a desired period of time (such as 24, 48 or 96 hours) in a 37 °C, 5% CO2 incubator.

For blank wells (medium without the cells), add the same amount of compound buffer. The suggested total volume is 100 µL for a 96-well plate, and 25 µL for a 384-well plate. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for proliferation or cytotoxicity induction. For proliferation assays, use fewer cells; for cytotoxicity assays, use more cells to start with.

Sample Protocol:

  1. Treat cells with test compounds as desired. Note: It is not necessary to wash cells before adding compound. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice after aspiration. Alternatively, cells can be grown in serum-free media.

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of dye-loading solution.

  3. Incubate the dye-loading plate at room temperature or 37°C for 1 hour, protected from light. (The incubation time could be from 15 minutes to overnight. We got the optimal results with the incubation time less than 4 hours.) Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. Note: DO NOT wash the cells after loading. Note: For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after incubation.

  4. Monitor the fluorescence intensity at Ex/Em = 490/525 nm with cutof=515 nm.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)494
Emission (nm)514

Citations


View all 17 citations: Citation Explorer
Functional imaging of neuronal activity of auditory cortex by using Cal-520 in anesthetized and awake mice
Authors: Li, Jingcheng and Zhang, Jianxiong and Wang, Meng and Pan, Junxia and Chen, Xiaowei and Liao, Xiang
Journal: Biomedical Optics Express (2017): 2599--2610
NINJ2--A novel regulator of endothelial inflammation and activation
Authors: Wang, Jingjing and Fa, Jingjing and Wang, Pengyun and Jia, Xinzhen and Peng, Huixin and Chen, Jing and Wang, Yifan and Wang, Chenhui and Chen, Qiuyun and Tu, Xin and others, undefined
Journal: Cellular Signalling (2017)
Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B
Authors: Bogert, Nicolai V and Werner, Isabella and Kornberger, Angela and Meybohm, Patrick and Moritz, Anton and Keller, Till and Stock, Ulrich A and Beiras-Fern, undefined and ez, Andres
Journal: Scientific reports (2016)
Inhibition of ABC transport proteins by oil sands process affected water
Authors: Alharbi, Hattan A and Saunders, David MV and Al-Mousa, Ahmed and Alcorn, Jane and Pereira, Alberto S and Martin, Jonathan W and Giesy, John P and Wiseman, Steve B
Journal: Aquatic Toxicology (2016): 81--88
Rapid generation of collagen-based microtissues to study cell--matrix interactions
Authors: Brett, Marie-Elena and Crampton, Alex and ra L , undefined and Wood, David K
Journal: Technology (2016): 1--8
Toxicokinetics and toxicodynamics of chlorpyrifos is altered in embryos of Japanese medaka exposed to oil sands process-affected water: evidence for inhibition of P-glycoprotein
Authors: Alharbi, Hattan A and Alcorn, Jane and Al-Mousa, Ahmed and Giesy, John P and Wiseman, Steve B
Journal: Journal of Applied Toxicology (2016)
Flexible Endoscopic Spray Application of Respiratory Epithelial Cells as Platform Technology to Apply Cells in Tubular Organs
Authors: Thiebes, Anja Lena and Reddemann, Manuel Armin and Palmer, Johannes and Kneer, Reinhold and Jockenhoevel, Stefan and Cornelissen, Christian Gabriel
Journal: Tissue Engineering Part C: Methods (2016): 322--331
Erythropoietin Stimulates Endothelial Progenitor Cells to Induce Endothelialization in an Aneurysm Neck After Coil Embolization by Modulating Vascular Endothelial Growth Factor
Authors: Liu, Peixi and Zhou, Yingjie and An, Qingzhu and Song, Yaying and Chen, Xi and Yang, Guo-Yuan and Zhu, Wei
Journal: MEDICINE (2016): 1--8
Spraying respiratory epithelial cells to coat tissue-engineered constructs
Authors: Thiebes, Anja Lena and Albers, Stefanie and Klopsch, Christian and Jockenhoevel, Stefan and Cornelissen, Christian G
Journal: BioResearch open access (2015): 278--287
Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions
Authors: Le Bihan, Olivier and Decossas, Marion and Gontier, Etienne and Gerbod-Giannone, Marie-Christine and Lambert, Olivier
Journal: Journal of structural biology (2015): 470--477

References


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Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development
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Journal: Biochemical and Biophysical Research Communications (2017)
Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
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A vaccination and challenge model using calcein marked fish
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Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
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Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake
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Calcein AM release-based cytotoxic cell assay for fish leucocytes
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Journal: Fish Shellfish Immunol (2004): 127
Calcein-AM is a detector of intracellular oxidative activity
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Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines
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In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue
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Application notes


Annexin V