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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: 1-541-465- 8300
2. Chinese Hamster Ovary (CHO) cells
3. 96 well black microtiter plates with clear bottoms (Costar, cat# 3603). Tel: 1-800-492- 1110
4. PBS (Phosphate buffered saline, 136.8mM NaCl, 2.5mM KCl, 0.8mM Na ₂ PO ₄ , 1.47mM KH ₂ PO ₄ , 0.9mM CaCl ₂ , 0.5mM MgCl ₂ pH 7.4)
5. 10% Triton X-100 in PBS
6. 0.526mM EDTA in PBS
7. Ham’s 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 Ham’s 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/100µl. 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/100µl. 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 100µl of PBS.

A solution of 11.4µM cal AM/5.7µM EthD-1 in PBS was made and 100µl aliquots were added to each well of the microtiter plate, including the control wells. The final concentration of dyes in each well was 5.7µM cal AM/2.85µM EthD-1. Cells were incubated with the dyes for 30 min at 37^o C 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.

Figure 1: Excitation and emission spectral scans of cal AM/EthD-1 stained cells. Scans were performed with PMT set to "auto", calibrate "on" and 20 reads per well.

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 10⁴ cells 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.

Table 1: Raw data from dilutions of live and dead cells stained with a combination of Cal AM and EthD-1. A, Ex485/ Em525; B, Ex525/Em620; Live*=live cells; dead=*dead cells; Std. Dev.=standard deviation; CV%=coefficient of variation

Figure 2: Differentiation of live and dead cells. Cal AM/EthD-1 stained cells were analyzed in well scan mode (fill pattern; 10 reads/well) under optimal Ex/Em and cutoff settings as described in the Results section. Mean RFU values were plotted against cell number with standard deviations indicated as error bars. A, shows the fluorescent signal (RFU) obtained with live (solid black circle) and dead (open circle) cells scanned to detect cal AM. B, shows the fluorescent signal obtained with live and dead cells scanned to detect EthD-1. These graphs demonstrate the difference in signal between live and dead cells.

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

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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).
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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).

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