CellQuanti-BlueTM Cell Viability Assay Kits

Non-radioactive Fluorescent Assay for Cell Proliferation and Cytotoxicity

This homogeneous assay involves simply adding a single reagent, the CellQuanti-BlueTM 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- BlueTM 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 reagentand 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 CellQuanti-BlueTM 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.

KEY FEATURES

Safe. Non-radioactive assay (cf. 3H-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-incubatemeasure" 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.

APPLICATIONS

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.

Control Reagent (Cat # CTTX-050): 50 mg saponin (to be ordered separately).

Storage conditions. The CellQuanti-BlueTM reagent is light sensitive and should be stored in the provided amber bottle at 4°C. The Control Reagent is stable at -20°C. Shelf life: 12 months.

This Protocol can be downloaded from www.bioassaysys.com.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

The CellQuanti-BlueTM assay is based on the conversion of the nonfluorescent 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.

Important: bring reagent to room temperature before use.

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 CellQuanti-BlueTM 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).

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 CellQuanti-BlueTM 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).

GENERAL CONSIDERATIONS

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 CellQuanti-BlueTM 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 CellQuanti-BlueTM 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.

The study of cell proliferation and cell viability requires the accurate quantification of the number of viable cells in a cell culture. Therefore, assays for calculating cell viability are necessary for optimizing cell culture conditions, evaluating cell growth factors and nutrients, discovering novel antibiotics and anti-cancer drugs, evaluating toxic effects of environmental pollutants and cell mediated toxicity and studying programmed cell death (apoptosis). The CellQuanti-MTTTM assay kit provides a convenient, sensitive, quantitative and reliable assay for determining the number of viable cells in a given culture. This homogeneous colorimetric assay is based on the conversion of a tetrazolium salt MTT, a pale yellow substrate, to formazan, a purple dye. This cellular reduction reaction involves the pyridine nucleotide cofactors NADH/NADPH and is only catalyzed by living cells. The formazan product has a low aqueous solubility and is present as purple crystals. Dissolving the resulting formazan with a solubilization buffer permits the convenient quantification of product formation. The intensity of the product color, measured at 550 - 620 nm, is directly proportional to the number of living cells in the culture. Reagents in the kit have been carefully formulated and optimized for sensitivity, assay robustness and automation.

KEY FEATURES

Safe. Non-radioactive assay (cf. 3H-thymidine incorporation assay).

Sensitive and accurate. As low as 950 cells can be accurately quantified.

Fast. High-throughput assay using 96-well plates allows simultaneous processing tens of thousands of samples per day.

Homogeneous and convenient. "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved.

Robust and amenable to HTS. Z’ factors of 0.5 and above are observed. Can be readily automated with HTS liquid handling systems.

APPLICATIONS

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 screen for toxic and anticancer drugs.

Control Reagent (Cat # CTTX-050): 50 mg saponin (to be ordered separately).

Storage conditions. Store the Reagent at -20 °C. Keep Assay Buffer and Solubilization Solution at 4 °C and room temperature, respectively. Shelf life: 12 month.

This protocol can be downloaded online at www.bioassaysys.com.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

The CellQuanti-MTTTM assay is based on the conversion of MTT to purple formazan by metabolically active cells. For most cells, this reducing reaction takes 3 to 5 hours. The produced formazan is solubilized and the resulting colored solution is quantified with a 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.

1. Plate and culture cells (80 μL per well) in a clear bottom 96-well tissue culture plates. Typical culture medium contains DMEM, 10% fetal bovine serum, antibiotics (penicillin/streptomycin, 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 1,000 to 80,000 per well. The volume can vary from 50 to 150 μL, although 80 μL is used in this example. In addition to the test samples, one must include control wells of culture medium containing no cells or cells treated with a toxic reagent such as 0.1% saponin.

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. A volume of 20 μL in phosphate buffered saline (PBS) or culture medium is recommended for the test compounds and controls. The Control reagent can be conveniently reconstituted with 5 mL PBS (1% saponin).

3. Reconstitute the CellQuanti-MTTTM Reagent with Assay Buffer. First equilibrate the Reagent and Assay Buffer to room temperature. Then simply combine Assay Buffer and the Reagent by pipetting a small volume (e.g. 1 mL) buffer to the Reagent tube. Vortex briefly and pipet the reconstituted solution to the Assay Buffer bottle. Repeat this step to transfer all Reagent to the Assay Buffer bottle. Mix by inversion until the Reagent is thoroughly dissolved. After this is done, mark the bottle label as Reconstituted Reagent. Note: Fresh reconstitution is recommended although the reconstituted reagent is stable for up to 4 weeks when stored at -20 °C.

4. Add 15 μL (per 80 μL cell culture) of CellQuanti-MTTTM reagent per well and incubate for 4 hours at 37°C. The volume of the reagent should be adjusted depending on the volume of cell culture.

5. Add 100 μL of the Solubilization Solution. Mix gently on an orbital shaker for one hour at room temperature. The volume of the Solubilization Solution should be adjusted depending on the volume of cell culture. If precipitation occurs in the Solubilization Solution, place the bottle in a warm water bath or at 37°C and shake to dissolve precipitates.

6. Measure OD570nm for each well on an absorbance plate reader. Maximum absorbance of the formazan dye lies between 560 and 590 nm. If desired, the OD measurement can be performed  the following day. In this case, it is recommended to seal the plate to minimize evaporation.

Determine the average of the blank controls and subtract this amount from all absorbance values. Plot the corrected absorbance values at 570nm against the concentration of the test compound. Determine the EC50 value for cell proliferation and IC50 value for cytotoxic compound by non-linear regression analysis using Prism or any other data analysis tools.

LITERATURE

Cell proliferation:

1. Luan X, Diekwisch TG (2002). CP27 affects viability, proliferation, attachment and gene expression in embryonic fibroblasts. Cell Prolif. 35: 207-19.

2. Bezivin C, Tomasi S, Lohezic-Le Devehat F, Boustie J (2003). Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine. 10(6-7):499-503.

3. Koh J, Kubota T, Koyama T, Migita T, Hashimoto M, Hosoda Y, Kitajima M (2000). Combined Antitumor Activity of 7- Hydroxystaurosporine (UCN-01) and Tamoxifen against Human Breast Carcinoma in vitro and in vivo. Breast Cancer. 10(3):260-7.

4. Labieniec M, Gabryelak T (2003). Effects of tannins on Chinese hamster cell line B14. Mutat Res. 539(1-2):127-35.

5. Camps J, About I (2003). Cytotoxicity testing of endodontic sealers: a new method. J Endod. 29(9):583-6.

KEY FEATURES

Safe. Non-radioactive assay (cf. 3H-thymidine incorporation assay).

Sensitive and accurate. As low as 950 cells can be accurately quantified.

Fast. High-throughput assay using 96-well plates allows simultaneous processing tens of thousands of samples per day.

Homogeneous and convenient. "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved.

Robust and amenable to HTS. Z’ factors of 0.5 and above are observed. Can be readily automated with HTS liquid handling systems.

APPLICATIONS

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 screen for toxic and anticancer drugs.

LITERATURE

Cell proliferation:

1. Luan X, Diekwisch TG (2002). CP27 affects viability, proliferation, attachment and gene expression in embryonic fibroblasts. Cell Prolif. 35: 207-19.

2. Bezivin C, Tomasi S, Lohezic-Le Devehat F, Boustie J (2003). Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine. 10(6-7):499-503.

3. Koh J, Kubota T, Koyama T, Migita T, Hashimoto M, Hosoda Y, Kitajima M (2000). Combined Antitumor Activity of 7- Hydroxystaurosporine (UCN-01) and Tamoxifen against Human Breast Carcinoma in vitro and in vivo. Breast Cancer. 10(3):260-7.

4. Labieniec M, Gabryelak T (2003). Effects of tannins on Chinese hamster cell line B14. Mutat Res. 539(1-2):127-35.

5. Camps J, About I (2003). Cytotoxicity testing of endodontic sealers: a new method. J Endod. 29(9):583-6.

Adenosine 5'-triphosphate (ATP) is the chemical energy for cellular metabolism and is often referred to as “energy currency" of the cell. ATP is produced only in living cells during photosynthesis and cellular respiration and consumed in cellular processes including biosynthetic reactions, motility and cell division. It is a key indicator of cellular activity and has been utilized as a measure of cell viability and cytotoxicity in research and drug discovery.

BioAssay Systems’ EnzyLightTM Cytotoxicity Assay Kit provides a rapid method to measure intracellular ATP, cell viability and cytotoxicity. The single working reagent lyses cells to release ATP, which, in the presence of luciferase, immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration and hence number of living cells.

KEY FEATURES

Safe. Non-radioactive assay (cf. 3H-thymidine incorporation assay).

Sensitive and accurate. As low as 50 cells can be quantified.

Homogeneous and convenient. "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved.

Robust and amenable to HTS: Z’ factors of 0.6 to 0.7 are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

APPLICATIONS

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.

KIT CONTENTS

Assay Buffer: 10 mL

Substrate: 120 μL

ATP Enzyme: 120 μL

Storage conditions: store all reagents at -20°C. Shelf life of at least 6 months.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

1. Cell Culture. Plate cells at 100 μL/well in white opaque tissue culture plates. If desired, add 5 μL test compounds and controls dissolved in PBS or culture medium per well. Rock plate lightly to mix and incubate for desired period of time (e.g. overnight).

2. Assay. Bring all components to room temperature. Keep thawed ATP Enzyme on ice or 4°C. For each test well, mix 95 μL Assay Buffer with 1 μL Substrate and 1 μL ATP Enzyme. Add 90 μL Reconstituted Reagent to each test well and mix by tapping the plate. Incubate for 2 minutes at room temperature. Read luminescence on a luminometer. For most luminometers (Berthold Luminometer, LJL Analyst, Top Count, MicroBeta Counters, CLIPR and LeadSeeker), integration time of 0.1 to 5 sec is appropriate.

1. Cell Culture. Plate cells at 25 μL/well in white opaque tissue culture plates. If desired, add 5 μL test compounds and controls dissolved in PBS or culture medium per well. Rock plate lightly to mix and incubate for desired period of time (e.g. overnight).

2. Assay. Bring all components to room temperature. Keep thawed ATP enzyme on ice or 4°C. For each test well, mix 30 μL Assay Buffer with 0.3 μL Substrate and 0.3 μL ATP Enzyme. Add 25 μL Reconstituted Reagent to each well and mix by tapping the plate. Incubate for 2 minutes at room temperature. Read luminescence on a luminometer. For most luminometers (Berthold Luminometer, LJL Analyst, Top Count, MicroBeta Counters, CLIPR and LeadSeeker), integration time of 0.1 to 5 sec is appropriate.

GENERAL CONSIDERATIONS

Signal stability. After adding the Reconstituted Reagent, the luminescence signal is stable for about 15 min and decreases slow thereafter. Reading is best performed within 30 min.

[1]. Li W. et al (2006). Human primary renal cells as a model for toxicity assessment of chemo-therapeutic drugs. Toxicol In Vitro. 20(5):669-76.

[2]. Zhelev Z, et al (2004). Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes. Phenothiazines and leukemia. Cancer Chemother Pharmacol. 53(3):267-75.

[3]. Ingram PR, et al (2004). A comparison of the effects of ocular preservatives on mammalian and microbial ATP and glutathione levels. Free Radic Res.