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Cell Meter™ Cell Viability Assay Kit *Red Fluorescence*

HeLa cell number response was measured with Cell Meter™ Cell Viability Assay Kit. HeLa cells at 0 to 3,000 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 CytoCalcein™ Red dye-working solution for 30 minutes at 37°C. The fluorescence intensity was measured at Ex/Em = 540/590 nm (Cutoff = 570 nm) with bottom read mode using Flexstation (from Molecular devices).
HeLa cell number response was measured with Cell Meter™ Cell Viability Assay Kit. HeLa cells at 0 to 3,000 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 CytoCalcein™ Red dye-working solution for 30 minutes at 37°C. The fluorescence intensity was measured at Ex/Em = 540/590 nm (Cutoff = 570 nm) with bottom read mode using Flexstation (from Molecular devices).
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Catalog Number22783
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InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


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 a proprietary non-fluorescent dye that gets enhanced red fluorescence upon entering into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the non-fluorescent substrate by intracellular esterases generates a strongly fluorescent hydrophilic product 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 the product generated from the esterase-catalyzed hydrolysis of the fluorogenic substrate. 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. The fluorogenic indicator has spectral properties compatible with Cy3/TRITC filter set.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode


Component A: CytoCalcein™ Red AM2 vials, lyophilized
Component B: DMSO1 vial (100 µL)
Component C: Assay Buffer1 bottle (20 mL)

Example protocol


Protocol summary

  1. Prepare cells with test compounds
  2. Remove the medium
  3. Add CytoCalcein™ Red AM working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  4. Incubate at room temperature or 37°C for 30 minutes - 1 hr
  5. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) 

Important notes
Thaw one of each kit component at room temperature before starting the experiment.


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™ Red AM stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Red AM (Component A) and mix well to make CytoCalcein™ Red AM stock solution. Note: 20 µL of CytoCalcein™ Red AM stock solution is enough for one plate. Protect from light. For storage, seal tubes tightly. Note: Unused CytoCalcein™ Red stock solution can be aliquoted and stored at < -20 oC for one month if the tubes are sealed tightly. Avoid repeated freeze-thaw cycles.


Add the whole content (20 µL) of CytoCalcein™ Red AM stock solution into 10 mL of Assay Buffer (Component C), and mix well to make CytoCalcein™ Red AM working solution. Note: This CytoCalcein™ Red AM working solution is not stable, use it promptly. 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. Aliquot and store the unused probenecid stock solution at ≤ -20 oC.

For guidelines on cell sample preparation, please visit


  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) and 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. Remove growth medium.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ Red AM working solution.

  4. Incubate plate at room temperature or 37°C for 30 minutes to 1 hour, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after incubation.

  5. Monitor the fluorescence intensity with a fluorescence plate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm).


View all 18 citations: Citation Explorer
Regulation of pancreatic stellate cell activation by Notch3
Authors: Song, Haiyan and Zhang, Yuxiang
Journal: BMC cancer (2018): 36
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


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Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development
Authors: Li, Deng and Xiang, Xiaocong and Yang, Fei and Xiao, Dongqin and Liu, Kang and Chen, Zhu and Zhang, Ruolan and Feng, Gang
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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Journal: Fish Shellfish Immunol (2006): 20
Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
Authors: Bratosin D, Mitrofan L, Palii C, Estaquier J, Montreuil J.
Journal: Cytometry A (2005): 78
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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Journal: Toxicol In Vitro (2005): 505
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
Authors: Mueller H, Kassack MU, Wiese M.
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In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue
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Journal: Biomaterials (2003): 3885
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Application notes

Annexin V