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Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit

CHO-K1 cell number response was measured with Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit. CHO-K1 cells at 0 to 10,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 20 µL/well of Assay Solution (Component A) for 3 hours at 37°C. The fluorescence intensity was measured at Ex/Em = 540/590 nm (Cutoff = 570 nm) with NOVOstar instrument (BMG Labtech). The fluorescence intensity was linear (R2 = 0.998) to the cell number as indicated.
CHO-K1 cell number response was measured with Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit. CHO-K1 cells at 0 to 10,000 cells/well/100 µL were seeded overnight in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 20 µL/well of Assay Solution (Component A) for 3 hours at 37°C. The fluorescence intensity was measured at Ex/Em = 540/590 nm (Cutoff = 570 nm) with NOVOstar instrument (BMG Labtech). The fluorescence intensity was linear (R2 = 0.998) to the cell number as indicated.
Ordering information
Price ()
Catalog Number22781
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
Extinction coefficient (cm -1 M -1)650001
Excitation (nm)571
Emission (nm)584
Quantum yield0.751
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Extinction coefficient (cm -1 M -1)
650001
Excitation (nm)
571
Emission (nm)
584
Quantum yield
0.751
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. The measurement of mitochondrial dehydrogenases (e.g. LDH) activity is a well-accepted assay to quantify cell numbers and monitor cell viability. This cell viability assay kit provides a fast, simple, accurate and homogeneous assay for the fluorometric detection of viable cells. This assay is based on the observation that blue and non-fluorescent resazurin is reduced to a pink fluorescent dye (resorufin) by accepting the electron from mitochondrial respiratory chain in live cells. The amount of resorufin produced is directly proportional to the number of living cells. The detection sensitivity of cell proliferation and cytotoxicity assays using this kit is higher than other assays such as MTT. Since the kit components are quite stable with minimal cytotoxicity, a longer incubation (such as 24 to 48 hours) is possible. The assay can be performed in a convenient 96-well and 384-well microtiter-plate format. The characteristics of its high sensitivity (<100 CHO cells), non-radioactive and no-wash method made the kit suitable for high throughput screening of cell proliferation or cytotoxicity against a variety of compounds and adaptable for a wide variety of instrument platforms. The kit provides all the essential components with an optimized assay protocol. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. Using 20 ul of reagents per well in a 96-well format, this kit provides sufficient reagents to perform 1000 assays. Using 10 ul of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 2,000 assays.

Platform


Fluorescence microplate reader

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

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (100 µL/well/96-well plate or 50 µL/well/384-well plate)
  2. Add 1/5 volume of Assay Solution (Component A)
  3. Incubate the cells in a 37°C, 5% CO2 incubator for 1 - 24 hours 
  4. Monitor fluorescence intenstity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw the component at room temperature before starting the experiment.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Plate 100 to 10,000 cells per 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 50 µL for a 384-well plate.

  2. Set up the following controls at the same time.
    1. Positive control: contains cells and known proliferation or cytotoxicity inducer.

    2. Negative control: contains cells but no test compounds.

    3. Vehicle control: contains cells and the vehicle used to deliver test compounds.

    4. Non-cell control: contains growth medium without cells. Note: LDH in serum will contribute to background fluorescence.

    5. Test compound control: contains the vehicle used to deliver test compounds [Hank’s balance salt solution (HBSS) or phosphate-buffered saline (PBS)] and test compound. Some test compounds have strong autofluorescence and may give false positive results. Note: Match the total volume of all the controls to 100 µL for a 96-well plate or 50 µL for a 384-well plate with growth medium.

  3. Warm up the Assay Solution (Component A) to 37°C, and mix it thoroughly before starting the experiments.

  4. Add 20 µL/well (96-well plate) or 10 µL/well (384-well plate) of Assay Solution (Component A) into each well. Mix the reagents by shaking the plate gently for 30 seconds.

  5. Incubate the cells in a 37°C, 5% CO2 incubator for 1 - 24 hours, protected from light. Note: The appropriate incubation time depends on the metabolism rate of the individual cell type and cell concentration used. Optimize the incubation time for each experiment. Extremely prolonged incubation time is not recommended since resazurin could be converted to colorless dihydroresorufin.

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

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)650001
Excitation (nm)571
Emission (nm)584
Quantum yield0.751

References


View all 65 references: Citation Explorer
Determination of the diagnostic value of the resazurin reduction assay for evaluating boar semen by receiver operating characteristic analysis
Authors: Zrimsek P, Kosec M, Kunc J, Mrkun J.
Journal: Asian J Androl (2006): 343
Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes
Authors: Rolon M, Vega C, Escario JA, Gomez-Barrio A.
Journal: Parasitol Res (2006): 103
Rapid susceptibility test for Mycobacterium tuberculosis to isoniazid and rifampin with resazurin method in screw-cap tubes
Authors: Coban AY, Cekic Cihan C, Bilgin K, Uzun M, Akgunes A, Cetinkaya E, Durupinar B.
Journal: J Chemother (2006): 140
A resazurin-based biosensor for organic pollutants
Authors: Tizzard AC, Bergsma JH, Lloyd-Jones G.
Journal: Biosens Bioelectron (2006): 759
Development and application of a resazurin-based biomass activity test for activated sludge plant management
Authors: McNicholl BP, McGrath JW, Quinn JP.
Journal: Water Res. (2006)
A fluorometric study of relative ocular lens cytosensitivity to multipurpose contact lens solutions using the resazurin assay method
Authors: Oriowo MO., undefined
Journal: Toxicol In Vitro (2006): 1548
Application of the resazurin microtitre assay for detection of multidrug resistance in Mycobacterium tuberculosis in Algiers
Authors: Nateche F, Martin A, Baraka S, Palomino JC, Khaled S, Portaels F.
Journal: J Med Microbiol (2006): 857
Evaluation of the resazurin microtiter assay for rapid detection of ofloxacin resistance in M. tuberculosis
Authors: Umubyeyi AN, Martin A, Zissis G, Struelens M, Karita E, Portaels F.
Journal: Int J Tuberc Lung Dis (2006): 808
Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis
Authors: Montoro E, Lemus D, Echemendia M, Martin A, Portaels F, Palomino JC.
Journal: J Antimicrob Chemother (2005): 500
The resazurin microplate method for rapid detection of vancomycin resistance in enterococci
Authors: Coban AY, Darka O, Fisgin NT, Cihan CC, Bilgin K, Akgunes A, Guven T, Dokuzoguz B, Birinci A, Durupinar B.
Journal: J Chemother (2005): 361