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Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence with 405 nm Excitation*

There are a variety of parameters that can be used to monitor cell viability. The proprietary violet laser -excitable fluorescent dye used in the kit is a hydrophobic compound that easily permeates intact live cells and gets enhanced fluorescence upon entering into live cells. The hydrolysis of the non-fluorescent substrate by intracellular esterases generates a strongly blue fluorescent product that is well-retained in the cell cytoplasm. The blue fluorophore generated by the esterase hydrolysis of the non-fluorescent substrate has the spectral properties of fluorescein. When excited at 405 nm, the fluoreophore emits intense blue fluorescence at ~450 nm. The kit provides all the essential components with an optimized cell-labeling protocol for fluorescence microplate assays. This Cell Meter™ Cell Viability Assay Kit provides an effective tool of labeling cells for fluorescence flow cytometry, microplate and microscopic investigations of cellular functions. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit is suitable for proliferating and non-proliferating cells.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds
  2. Add the same volume of CytoCalcein™ Violet 450, AM working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  3. Incubate at room temperature or 37°C for 1 hour
  4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 405/460 nm (Cutoff = 435 nm)

Important notes
Thaw one of each kit component 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. CytoCalcein™ Violet 450, AM stock solution:
Add 20 µL of DMSO (Component B) into the vial of CytoCalcein™ Violet 450, AM (Component A), and mix well to make CytoCalcein™ Violet 450, AM stock solution. Note: 20 µL of CytoCalcein™ Violet 450, AM stock solution is enough for one plate. Protect from light. For storage, seal tubes tightly.

PREPARATION OF WORKING SOLUTION

Add the whole content (20 µL) of CytoCalcein™ Violet 450, AM stock solution into 10 mL of Assay Buffer (Component C), and mix well to make CytoCalcein™ Violet 450, AM working solution. This CytoCalcein™ Violet 450, AM 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 promote 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 ranging from 1 to 2.5 mM. Aliquot and store the unused probenecid stock solution at ≤ -20 °C.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat cells with test compounds as desired. Note: It is not necessary to wash cells before adding compounds. 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 a serum-free media.

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ Violet 450, AM working solution.

  3. Incubate the 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 with a fluorescence plate reader (bottom read mode) at Ex/Em = 405/460 nm (Cutoff = 435 nm).

Spectrum

Product family

Citations

View all 18 citations: Citation Explorer
Regulation of high glucose-induced apoptosis of brain pericytes by mitochondrial CA VA: A specific target for prevention of diabetic cerebrovascular pathology
Authors: Price, Tulin O and Sheibani, Nader and Shah, Gul N
Journal: Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease (2017): 929--935
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

References

View all 84 references: Citation Explorer
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
Authors: Zibek S, Stett A, Koltay P, Hu M, Zengerle R, Nisch W, Stelzle M.
Journal: Biophys J. (2006)
A vaccination and challenge model using calcein marked fish
Authors: Klesius PH, Evans JJ, Shoemaker CA, Pasnik DJ.
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
Authors: Schoonen WG, Westerink WM, de Roos JA, Debiton E.
Journal: Toxicol In Vitro (2005): 505
Page updated on October 11, 2024

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Catalog Number22784
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Spectral properties

Excitation (nm)

406

Emission (nm)

445

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation405 nm
Emission460 nm
Cutoff435 nm
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode

Components

CHO-K1 cell number response was measured with Cell Meter&trade; Cell Viability Assay Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 &micro;L were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 &micro;L/well of CytoCalcein&trade; Violet 450, AM dye-working solution for 1 hour at room temperature. The fluorescence intensity was measured at Ex/Em = 405/460 nm with NOVOstar instrument (from BMG Labtech). The fluorescence intensity was linear (R<sup>2</sup> = 1) to the cell number as indicated.
CHO-K1 cell number response was measured with Cell Meter&trade; Cell Viability Assay Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 &micro;L were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 &micro;L/well of CytoCalcein&trade; Violet 450, AM dye-working solution for 1 hour at room temperature. The fluorescence intensity was measured at Ex/Em = 405/460 nm with NOVOstar instrument (from BMG Labtech). The fluorescence intensity was linear (R<sup>2</sup> = 1) to the cell number as indicated.
CHO-K1 cell number response was measured with Cell Meter&trade; Cell Viability Assay Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 &micro;L were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 &micro;L/well of CytoCalcein&trade; Violet 450, AM dye-working solution for 1 hour at room temperature. The fluorescence intensity was measured at Ex/Em = 405/460 nm with NOVOstar instrument (from BMG Labtech). The fluorescence intensity was linear (R<sup>2</sup> = 1) to the cell number as indicated.