Cell Viability Assay Kits

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Cellular Assay (CA)
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Characteristics Safe. Non-radioactive assay (cf. 3 H-thymidine incorporation assay).
Sensitive and accurate. As low as 100 cells can be accurately quantified.
Time efficient.
High-throughput assay in 96-well and 384-well plates allows simultaneous processing ten of thousands of samples per day.
Homogeneous and convenient. A single reagent and mix-incubate- measure type assay. No wash and reagent transfer steps are involved.
Robust and amenable to HTS: Z' factors of 0.6 to 0.9 are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.
Background Homogeneous assay for cell viability, proliferation, cytotoxcity, high-throµghput screening for anticancer agents. Fluorimetric method (530nm/590nm).

This homogeneous assay involves simply adding a single reagent, the reagent, to the cell culture and measuring the fluorescence intensity (excitation wavelength = 530 - 570 nm, emission wavelength = 590 - 620 nm) after an incubation step. The CellQuanti- Blue TM reagent, like other resazurin-based assays such as the Alamar Blue reagent, utilizes the redox dye resazurin which is not fluorescent, but upon reduction by metabolically active cells is converted into a highly fluorescent product (resorufin). Living cells can readily reduce this non-toxic reagent and the resulting increase in fluorescence intensity can be conveniently monitored using a fluorescence spectrophotometer or plate reader. Nonviable cells have no metabolic capacity and, thus, will not reduce the dye. Therefore, the fluorescence intensity observed in this assay is a true measure of the viable cells. The reagent has been optimized for maximum sensitivity, reproducibility and long shelf-life. The homogeneous cell-based assay can be performed in multi-well plates. The reagent is compatible with all culture media and with all liquid handling systems for high-throughput screening applications in 96-well and 384-well plates. Applications include cell proliferation, cytotoxicity and apoptosis.
Research Area Cell Viability, Proliferation
Application Notes Cell Proliferation: effects of cytokines, growth factor, nutrients.
Cytotoxicity and Apoptosis: evaluation of toxic compounds, anti-cancer antibodies, toxins, environmental pollutants etc.
Drug Discovery: high-throughput screening for anticancer drugs.
Comment

Incubation time. The incubation time is dependent on the cell line. Some cell lines exhibit strong metabolic activity and, thus, require shorter incubation time than less metabolically active cell lines. The incubation time can be easily determined by reading the plate multiple times e.g. every 30 minutes after adding the reagent. In general, incubation for 1 to 5 hours is sufficient. Extensive incubation (such as >18 hours) may result in non-linear fluorescence response at high cell numbers.
Cell number. Generally the optimized reagent shows a broad range linear fluorescence response to the number of culturing cells. It is recommended to determine the number of cells per well that gives a highest signal:noise ratio. The optimal cell number can be easily determined by serial dilution of cells.
Controls. A positive control that is either cytotoxic or promotes cell proliferation can be run although it is not required. Saponin is a cytotoxic detergent that is available from BioAssay Systems (see Figure 2 in Technical Notes). A blank control, i.e., culture medium without cells or cells containing 0.1% saponin, should be done for each assay. The blank control determines background fluorescence that must be subtracted for data analysis.

Protocol The assay is based on the conversion of the non-fluorescent reagent to fluorescent product by metabolically active cells. For most cells this reductive reaction takes 1 to 5 hours. The fluorescence intensity of the product is then quantified on a fluorescent microplate reader. Although most culture media contain phenol red, phenol red does not interfere with the assay. All data in Technical Notes were obtained in culture media containing phenol red.

Procedure using 96-well plate:
1. Plate and culture cells (80 µL) in black 96-well tissue culture plates. Typical culture medium contains DMEM, 10% fetal bovine serum and antibiotics (penicillin/ strepto-mycin, gentamycin, etc), amino acids and other nutrients. Assays can be performed on either adherent cells or cells in suspension. The number of cells can vary from 100 to 80,000 per well. The volume can vary from 50 to 150 µL, although 80 µL is used in this protocol. In addition to the test samples, control wells of culture medium containing no cells or cells treated with a toxic reagent such as 0.1% saponin should be included.
2. Add test compounds and controls and incubate cells for the desired period of time (typically overnight). It is recommended that assays be run in duplicate or triplicate. Compounds and controls (20 µL) can be added in phosphate buffered saline (PBS) or culture medium. The Control reagent can be conveniently reconstituted with 5 mL PBS (1% saponin).
3. Equilibrate the Reagent to room temperature. Add 10 µL (per 100 µL of cell culture) of the reagent per well. The volume of the reagent can be adjusted depending on the volume of cell culture. Tap plate to mix cells with compounds. Incubate for 1 to 5 hours at 37°C.
4. Measure fluorescent intensity for each well on a fluorescence plate reader. If a Molecular Devices LJL Analyst is used, use the rhodamine filter sets (530nm excitation filter, 590nm emission filter and 570nm dichroic mirror).

Procedure using 384-well plate:
1. Plate and culture cells (40 µL) in black 384-well tissue culture plates. The number of cells can vary from 100 to 20,000 per well. The volume can vary from 25 to 60 µL, although 40 µL is used in this protocol. In addition to the test samples, control wells of culture medium containing no cells or cells treated with a toxic reagent such as 0.1% saponin should be included.
2. Add test compounds and controls and incubate cells for the desired period of time. It is recommended that assays be run in duplicate or triplicate and that compounds be added in PBS or culture medium with a volume of 10 µL.
3. Equilibrate Reagent to room temperature. Add 5 µL Reagent (per 50 µL of cell culture). Tap plate lightly to mix Reagent with cells. Incubate for 1 to 5 hours at 37°C.
4. Measure fluorescent intensity for each well on a fluorescence plate reader. If a Molecular Devices LJL Analyst is used, use the rhodamine filter sets (530nm excitation filter, 590nm emission filter and 570nm dichroic mirror).
Reagent Preparation

Important: bring reagent to room temperature before use.

Restrictions For Research Use only
Storage 4 °C
Supplier Images
Cellular Assay (CA) image for Cell Viability Assay Kits (ABIN1000238) Cell Viability Assay Kits
Product cited in: Ramirez, Antczak, Djaballah: "Cell viability assessment: toward content-rich platforms." in: Expert opinion on drug discovery, Vol. 5, Issue 3, pp. 223-33, 2012 (PubMed).

Hemendinger, Armstrong, Brooks: "Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death." in: Toxicology and applied pharmacology, Vol. 251, Issue 3, pp. 217-25, 2011 (PubMed).

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Martino, Perea, Hopfner, Ferguson, Wintermantel: "Effects of weak static magnetic fields on endothelial cells." in: Bioelectromagnetics, Vol. 31, Issue 4, pp. 296-301, 2010 (PubMed).

Kelley, Miller, Todd, Kelley, Tuttle, Berberich: "YPEL3, a p53-regulated gene that induces cellular senescence." in: Cancer research, Vol. 70, Issue 9, pp. 3566-75, 2010 (PubMed).

Wang, Lee, Johnson, Allie, Hu, Calabresi, Nath: "Activated T-cells inhibit neurogenesis by releasing granzyme B: rescue by Kv1.3 blockers." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 30, Issue 14, pp. 5020-7, 2010 (PubMed).

Nakajima, Shimazawa, Mishima, Hara: "Neuroprotective effects of Brazilian green propolis and its main constituents against oxygen-glucose deprivation stress, with a gene-expression analysis." in: Phytotherapy research : PTR, Vol. 23, Issue 10, pp. 1431-8, 2009 (PubMed).

Zayas-Santiago, Kang Derwent: "Preservation of intact adult rat photoreceptors in vitro: study of dissociation techniques and the effect of light." in: Molecular vision, Vol. 15, pp. 1-9, 2009 (PubMed).

Mochida, Matsura, Yamashita, Horie, Ohata, Kusumoto, Nishida, Minami, Inagaki, Ishibe, Nakada, Ohta, Yamada: "Geranylgeranylacetone ameliorates inflammatory response to lipopolysaccharide (LPS) in murine macrophages: inhibition of LPS binding to the cell surface." in: Journal of clinical biochemistry and nutrition, Vol. 41, Issue 2, pp. 115-23, 2008 (PubMed).

Hemendinger, Armstrong, Persinski, Todd, Mougeot, Volvovitz, Rosenfeld: "Huperzine A provides neuroprotection against several cell death inducers using in vitro model systems of motor neuron cell death." in: Neurotoxicity research, Vol. 13, Issue 1, pp. 49-61, 2008 (PubMed).

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Background publications Kreisler, Christoffers, Willershausen, dHoedt: "Low-level 809 nm GaAlAs laser irradiation increases the proliferation rate of human laryngeal carcinoma cells in vitro." in: Lasers in medical science, Vol. 18, Issue 2, pp. 100-3, 2003 (PubMed).

Nordling, Glinghammar, Karlsson, de Kok, Rafter: "Effects on cell proliferation, activator protein-1 and genotoxicity by fecal water from patients with colorectal adenomas." in: Scandinavian journal of gastroenterology, Vol. 38, Issue 5, pp. 549-55, 2003 (PubMed).

Kipshidze, Moussa, Nikolaychik, Chekanov, Khanna, Colombo, Leon, Moses: "Influence of Class I interferons on performance of vascular cells on stent material in vitro." in: Cardiovascular radiation medicine, Vol. 3, Issue 2, pp. 82-90, 2003 (PubMed).

Gloeckner, Jonuleit, Lemke: "Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue." in: Journal of immunological methods, Vol. 252, Issue 1-2, pp. 131-8, 2001 (PubMed).

Mikus, Steverding: "A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue." in: Parasitology international, Vol. 48, Issue 3, pp. 265-9, 2001 (PubMed).

Byth, Mchunu, Dubery, Bornman: "Assessment of a simple, non-toxic Alamar blue cell survival assay to monitor tomato cell viability." in: Phytochemical analysis : PCA, Vol. 12, Issue 5, pp. 340-6, 2001 (PubMed).

Lee, Kim, Kim, Kim: "In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants." in: Toxicology in vitro : an international journal published in association with BIBRA, Vol. 14, Issue 4, pp. 345-9, 2000 (PubMed).

Nociari, Shalev, Benias, Russo: "A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity." in: Journal of immunological methods, Vol. 213, Issue 2, pp. 157-67, 1998 (PubMed).