Navigation Links
LIVE/DEAD Viability/Cytotoxicity Assay for Animal Cells Using the SpectraMax Gemini XS Fluorescence Microplate Reader (MaxLine Application Note #43)

Anne T. Ferguson, Ph.D.
Molecular Devices Corporation


INTRODUCTION
This application note describes the use of the SPECTRAmax GEMINI XS from Molecular Devices to study the performance of the LIVE/DEAD Viability/Cytoxicity Assay Kit for animal cells. This assay uses two fluorescent dyes, calcein AM (cal AM) and ethidium homodimer (EthD-1), to stain live and dead cells simultaneously. Cal AM is an electrically neutral, nonfluorescent, esterase substrate that diffuses into live cells and becomes enzymatically cleaved by ubiquitous cytoplasmic esterases. This releases the free calcein fluorophore that is retained inside live cells. The dye emits a strong green fluorescence that peaks at ~525nm when excited at ~485nm. In contrast, EthD-1 is a polar nucleic acid stain that can penetrate dead, but not live cell membranes. Once intercalated into nucleic acids, it produces a 40-fold increase in red fluorescence at ~625 when excited at ~525.

The present study first determines the optimal excitation (Ex) and emission (Em) wavelengths for each dye. Next, these Ex and Em wavelengths are used to determine how well the two dyes are distinguished in pure populations of live and dead cells in order to predict interference when both dyes are present.

LIVE/DEAD Viability/Cytotoxicity is a sensitive assay designed to monitor changes in the ratio of live to dead cells in the total cell population. Consequently, it is useful for screening compounds that induce cell death, as well as a basic research tool for studying apoptosis, cell-mediated immunity and cell proliferation (1-7). Some advantages of LIVE/DEAD Viability/Cytotoxicity are that live cell staining is not dependent on cell metabolism or proliferation as in the MTT and thymidine uptake assays, and it is a nonradioactive assay unlike thymidine uptake and 51Cr release assays (8, 9).


MATERIALS
1. LIVE/DEAD Viability/Cytoxicity Kit (Molecular Probes, cat# L-3224) Tel: 1-541465-8300

2. Chinese Hamster Ovary (CHO) cells

3. 96 well black microtiter plates with clear bottoms (Costar, cat# 3603). Tel: 1-800-4921110

4. PBS (Phosphate buffered saline, 136.8mM NaCl, 2.5mM KCl, 0.8mM Na2PO4, 1.47mM KH2PO4, 0.9mM CaCl2, 0.5mM MgCl2 pH 7.4)

5. 10% Triton X-100 in PBS

6. 0.526mM EDTA in PBS

7. Hams F12 medium Cat# 9058, Fetal Bovine Serum (FBS) Cat# 3000 (Irvine Scientific) Tel: 1-800-437-5706


METHODS
Cell Preparation
CHO cells were grown in Hams F12 medium containing 10% FBS. Cells were washed three times with PBS and then treated for 20 minutes with 3 mls of 0.526mM EDTA in PBS. The detached cells were harvested and diluted in PBS. An aliquot was counted using a hemocytometer.

The concentration of cells was adjusted to 2.5 x 105 cells/ml in PBS, which is equivalent to a starting concentration of 2.5 x 104 cells/100l. For preparation of dead cells, half of the cells were treated with 1:100 dilution of 10% Triton X-100 for 10 min. Both live and dead cells were serially diluted 1:3 in PBS to obtain a range of concentrations from 2.5 x 104 to 103 cells/100l. Cells were plated such that each column of the microplate had 8 replicates of a live or dead cell dilution. The negative control/background was 100l of PBS.

A solution of 11.4M cal AM/5.7M EthD-1 in PBS was made and 100l aliquots were added to each well of the microtiter plate, including the control wells. The final concentration of dyes in each well was 5.7M cal AM/2.85M EthD-1. Cells were incubated with the dyes for 30 min at 37oC in a tissue culture incubator and then analyzed using the GEMINI XS.


RESULTS
Excitation and Emission Wavelength Optimization
The parts of the plates containing 2.5 x 104 live and dead cells/well were scanned on spectrum mode to determine optimal Ex and Em wavelengths for the cal AM dye, as shown in Figure 1A. Next, this part of the plate was scanned to optimize wavelengths for the EthD-1 dye (Fig. 1B). The combination of Ex/Em wavelengths that showed the greatest difference between live and dead cells was chosen for future experiments. These include Ex485nm/Em525nm using a 515nm cutoff filter for cal AM stained live cells, and Ex525nm/Em620nm using a 590nm cutoff filter for EthD-1 stained dead cells. These settings were used for characterizing the performance of the assay.


Assay Performance
Plates were read using the well scan feature, taking readings at ten different positions in each well. The signal to background ratios were calculated by dividing the RFU value obtained for 2.5 x 104cells by the value for the negative control (PBS only). The values for cal AM and EthD-1 were 6.2 and 8.1, respectively. All of the wells containing live cells, dead cells and PBS only were scanned under optimal Ex/Em conditions for each dye to determine the linearity (Tables 1A, B). The CV% for all cell dilutions was well below 10%. The linear curve for live cells was compared to that obtained with the same number of dead cells, both were scanned at 485/525 (optimal for cal AM) (Fig. 2A). In this scan, the background signal for dead cells remained constant for up to the maximum, 2.5 x 104 cells/well. In addition, live and dead cell fluorescence was compared at 525/620 (Fig. 2B). In contrast, there was a small increase in signal from 103 to 2.5 x 104 live cells/well. In other words, 2.5 x 104 live cells/well produce a fluorescent signal approximately equal to 2.5 x 103 dead cells/well. This may be a result of fluorescence interference or a small (10%) number of contaminating dead cells within the live cell population. These two figures demonstrate that there is a significant difference in fluorescent signal between live and dead cells that easily differentiates the two cell populations when they are stained with a mixure of the dyes.



CONCLUSION
The LIVE/DEAD Viability/Cytoxicity assay is a simple and fast method for detection of live and dead cells. Using the optimized wavelength settings, the SPECTRAmax GEMINI XS fluorescence microplate reader can distinguish between live and dead cells using this assay. The data suggests that there is little interference from dead cells when live cells are analyzed, however, there may be some interference from live cells when dead cells are analyzed. This assay should be especially suitable for growth inhibition and cell death assays that emphasize determination of a relative decrease in live cell number using cal AM dye. A standard curve for both live and dead cells is easily generated using the SOFTmax PRO software and its integrated formulas.


REFERENCES
1. Wagner, M. M., Paul, D. C., Shih, C., Jordan, M. A., Wilson, L., and Williams, D. C. In vitro pharmacology of cryptophycin 52 (LY355703) in human tumor cell lines. Cancer Chemother. Pharmacol. 43: 115-125 (1999).

2. Adler, M., Shafer, H., Hamilton, T., and Petrali, J. P. Cytotoxic actions of the heavy metal chelator TPEN on NG108-15 neuroblastoma-glioma cells. Neurotoxicity 20: 571-582 (1999).

3. Papadopoulos, N. G., Dedoussis, G. V., Spanakos, G., Gritzapis, A. D., Baxevanis, C. N., and Papamichail, M. An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry. J. Immunol. Methods 177: 101111 (1994).

4. Wang, X. M., Terasaki, P. I., Rankin, G. W., Chia, D., Zhong, H. P., and Hardy, S. A new microcellular cytotoxicity test based on calcein AM release. Human Immunology 37: 264-270 (1993).

5. Hausner, J. M. L., Buehrer, B. M., and Bell, R. M. Role of ceramide in mitogenesis induced by exogenous sphingoid bases. J. Biol. Chem. 269: 6803-6809 (1994).

6. Jacobson, M. D., Weil, M. and Raff, M. C. Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J. Cell Biology 133: 1041-1051 (1996).

7. Weil, M., Jacobson, M. D., Coles, H. S. R., Davies, T. J., Gardner, R. L., Raff, K. D., and Raff, M. C. Constitutive expression of the machinery for programmed cell death. J. Cell Biology 133: 1053-1059 (1996).

8. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63 (1983)

9. Oral, H. B., George, A. J., and Haskard, D. O. A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury. Endothelium 6: 143-151 (1998).


back to top


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
2. Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" )
3. Organize Your Data from the Big Blue Mutation Assay
4. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
5. RNase Protection Assay (RPA) Using DIG-Labeled RNA Probes
6. measuring cell proliferation with SpectraMax M5, M2 and Gemini microplate readers using the CyQUANT Cell Proliferation Assay
7. Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success
8. Using Validated siRNAs in Functional Genomic Assays
9. Nuclease Protection Assays: The Basics
10. RNArticles: RNA Amplification Used to Develop a New In Vitro HCMV Assay
11. Detection of Phosphorylated and Total ERK and p38 MAPK by Bio-Plex Assay, ELISA, and Western Blotting, Rev A
Post Your Comments:
(Date:8/28/2014)... 2014 Best Sanitizers, Inc., the first ... hand sanitizer, asks food processors and food handling professionals ... sanitizer they’re currently using to Best Sanitizers’ Alpet® ... critical to fighting cross-contamination and the spread of pathogens ... there are key criteria that make a hand sanitizer ...
(Date:8/28/2014)... , Aug. 28, 2014 /PRNewswire/ ... new market research report is ... Whole Exome Sequencing Market ... Preparation, Target Enrichment), by Services ... Synthesis), by Application (Cancer, Monogenic ...
(Date:8/28/2014)... (PRWEB) August 28, 2014 Sterlitech is ... polymeric membranes to its stable of products . ... molecular weight cut-offs and include membrane filters with surface ... increase in customer demand for our membrane process testing ... variety of flat sheet membranes,” explains Sterlitech President Mark ...
(Date:8/28/2014)... (PRWEB) August 28, 2014 A ... around the world is available from Strategic Consulting, ... companies. “ Industrial Microbiology Market Review, Fourth Edition: ... Market ” (IMMR—4) tracks and compares microbiology test ... North America, Europe and Asia, and forecasts future ...
Breaking Biology Technology:Food Processors and Food Handlers are Encouraged to Prepare for Fall Harvest by Evaluating Their Current Hand Sanitizer Using Nine Key Criteria 2Food Processors and Food Handlers are Encouraged to Prepare for Fall Harvest by Evaluating Their Current Hand Sanitizer Using Nine Key Criteria 3Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 2Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 3Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 4Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 5Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 6Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 7Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 8Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 9Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 10Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 11Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 12Whole Exome Sequencing Market by Product (Systems, Kits (Library Preparation, Target Enrichment), by Services (Sequencing), by Technology (Sequencing by Synthesis), by Application (Cancer, Monogenic disorders) - Global Forecast to 2018 13Sterlitech Corporation Enhances Its Selection of Flat Sheet Membrane Filters 22 Billion Industrial Microbiology Tests Conducted for Product Quality and Safety Worldwide 22 Billion Industrial Microbiology Tests Conducted for Product Quality and Safety Worldwide 3
... palm plantations will worsen the dual environmental crises of ... protected, warn scientists in the most comprehensive review of ... from the Zoological Society of London and University of ... the role of palm oil production in tropical deforestation ...
... Micromet, Inc.,(Nasdaq: MITI ) ("Micromet" or ... for the treatment of cancer,inflammation and autoimmune diseases, ... board of directors appointed Dr. Peter Johann, a,current ... member of the Company,s,audit committee. The appointment fills ...
... 15 Inverness Medical,Innovations, Inc. (Amex: IMA ), ... solutions, today declared a,dividend of $4.77 per share on ... dividend will be paid in shares of Series B,stock ... of (a) $4.77 divided by,(b) 97% of the average ...
Cached Biology Technology:Oil palm plantations are no substitute for tropical rainforests, a new study shows 2Oil palm plantations are no substitute for tropical rainforests, a new study shows 3Micromet, Inc. Fills Audit Committee Vacancy and Regains Compliance With NASDAQ Marketplace Rule 2Micromet, Inc. Fills Audit Committee Vacancy and Regains Compliance With NASDAQ Marketplace Rule 3Micromet, Inc. Fills Audit Committee Vacancy and Regains Compliance With NASDAQ Marketplace Rule 4Inverness Medical Innovations Declares Dividend on its Series B Convertible Perpetual Preferred Stock 2
(Date:8/31/2014)... new study of satellite data from the last 19 years ... sea-level around the coast of Antarctica to rise by 2cm ... the University of Southampton detected the rapid rise in sea-level ... than a million square kilometres. , The melting ... ice shelves has contributed an excess of around 350 gigatonnes ...
(Date:8/31/2014)... Over the past several decades, malaria diagnosis has changed very ... technician smears the blood across a glass slide, stains it ... the Plasmodium parasite, which causes the disease. This approach gives ... blood an important measure of disease severity but ... , A research team from the Singapore-MIT Alliance for Research ...
(Date:8/29/2014)... some surprising discoveries about the body,s initial responses ... the team found that specialized cells in the ... viral invasion and are the source of gut ... (IL-1β). , Though aimed at the presence ... epithelium that provides a barrier to protect the ...
Breaking Biology News(10 mins):A new way to diagnose malaria 2A new way to diagnose malaria 3The early cost of HIV 2
... of Iowa study suggests there are two root causes of ... findings, which already have sparked a clinical trial, may guide ... patients with CF. Almost half of patients with ... quarter will develop it in their teens. In addition to ...
... a global life sciences technology company specializing in clinical ... latest version of iMedNet ™ ... is a ground-breaking eClinical solution that makes electronic data ... research initiative...even very small studies of short duration.  This ...
... HOUSTON -- (Sept. 17, 2012) -- In the largest ... research institutions are embarking upon a five-year National Institutes ... and more effective by uncovering the exact causes of ... is a life-giver, but hemoglobin can become deadly when ...
Cached Biology News:Cystic fibrosis disrupts pancreas two ways in CF-related diabetes 2Cystic fibrosis disrupts pancreas two ways in CF-related diabetes 3MedNet Solutions Announces Latest Enhancements To Its iMedNet EDC eClinical Solution 2Rice to take part in $10.8 million study of hemoglobin toxicity 2Rice to take part in $10.8 million study of hemoglobin toxicity 3
Purified anti-LAT...
Nuclear/Cytosol Fractionation Kit Research Focus: other Storage Temperature: 4C Shipping Temperature: 4C...
Cytosol/Particulate Rapid Separation Kit Research Focus: other Storage Temperature: -20C Shipping Temperature: 4C...
Request Info...
Biology Products: