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Gain More Informative Data by Multiplexing a Fluorescent Real-Time Cytotoxicity Assay...

Gain More Informative Data by Multiplexing a Fluorescent Real-Time Cytotoxicity Assay with Luminescent, Fluorescent or Colorimetric Viability Assays
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Abstract

The CellTox™ Green Cytotoxicity Assay can measure cytotoxicity kinetically in the same assay plate instead of requiring parallel plates, reducing unnecessary duplication of effort required for endpoint methods. Multiplexing assays gathers more than one set of data from the same sample. In this report, we show that the CellTox™ Green Cytotoxicity Assay is compatible with the CellTiter-Glo® Luminescent Cell Viability, CellTiter-Fluor™ Cell Viability and CellTiter 96® AQueous One Solution Cell Proliferation assays, allowing users to measure viability and cytotoxicity from the same sample.

Brad Hook, Mark Bratz and Trista Schagat

Promega Corporation

Publication Date: June 2013

Introduction

The CellTox™ Green Cytotoxicity Assay measures changes in membrane integrity that occur due to cell death (1) . The assay is intended for assessing cytotoxicity in cell culture after experimental manipulation. The assay system uses an asymmetric cyanine dye that is excluded from viable cells but preferentially stains the DNA in dead cells (Figure 1, Panel A). When the dye binds DNA in compromised cells, its fluorescence properties are substantially enhanced. Viable cells produce no appreciable increase in fluorescence; therefore, the fluorescence signal produced by the binding to dead cell DNA is proportional to cytotoxicity. The dye can be diluted in culture medium and delivered directly to cells at the time of seeding or when compound is added, allowing no-step kinetic measures of cytotoxicity (Figure 1, Panel B). The dye can also be diluted in assay buffer and delivered to cells as a conventional endpoint measure (Figure 1, Panel B). This is a stable system to use because it is not based on protein biomarkers that can degrade during the course of treatment and lead to signal loss. DNA is more stable and therefore gives a steadier, longer lasting signal than other current cytotoxicity chemistries.

Figure 1. CellTox™ Green Cytotoxicity Assay.

Panel A. CellTox™ Green Dye binds DNA of cells with impaired membrane integrity. Panel B. Schematic diagram of methods for delivering the CellTox™ Green Dye.

Panel A. CellTox™ Green Dye binds DNA of cells with impaired membrane integrity. Panel B. Schematic diagram of methods for delivering the CellTox™ Green Dye.

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Multiplexing assays gathers more than one set of data from the same sample. The key benefit of multiplexing is gaining a better understanding of the event you are measuring in the context of another parameter, minimizing faulty interpretation of data or ambiguity from data sets. Multiplexing insures that the variables for a sample are the same for all the assays and new variables are not introduced through replicate plating. Multiplexing also provides a way to normalize data to a particular parameter; for instance, coupling the ratio of an activity to the number of cells in the well. To multiplex two or more assays, the assays must meet certain criteria: the signals for the various assays must be spectrally or temporally distinct, the assay chemistries must be compatible, and the assays must fit into the same well or be easily separated (e.g., moving medium to a separate plate and performing further experiments on the cells).

In this report, we show that the CellTox™ Green Cytotoxicity Assay can be multiplexed with the bioluminescent-based CellTiter-Glo® Luminescent Viability Assay, the fluorescent-based CellTiter-Fluor™ Cell Viability Assay and the absorbance-based CellTiter 96® AQueous One Solution Cell Proliferation Assay. The CellTiter-Glo® Luminescent Cell Viability Assay determines the number of viable cells in culture based on ATP quantitation, which signals the presence of metabolically active cells. The CellTiter-Fluor™ Cell Viability Assay measures the activity of a conserved, constitutive protease within live cells and therefore, serves as a marker of cell viability. The CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells through bioreduction of a MTS tetrazolium compound. Multiplexing CellTox™ Green Cytotoxicity Assay with these viability assays assesses both kinetic measurement of cytotoxicity and endpoint cell viability.

Materials and Methods

Assays CellTox™ Green Cytotoxicity Assay (Cat.# G8741)
CellTiter-Glo® Luminescent Viability Assay (Cat.# G7570)
CellTiter-Fluor™ Cell Viability Assay (Cat.# G6080)
CellTiter 96® AQueous One Solution Cell Proliferation Assay (Cat.# G3582)
Cultured Cells HEK 293 cells cultured in DMEM + 10% FBS
Note: Cells have been authenticated using the GenePrint® 10 System (Cat.# B9510).
Compounds terfenadine (Sigma Cat.# T9652)
ionomycin (Sigma Cat.# I3909)
Equipment and Supplies white 96-well tissue culture treated plates (Costar Cat.# 3917)
black 96-well tissue culture treated plates (Costar Cat.# 3916)
black 96-well clear-bottom tissue culture treated plates (Costar Cat.# 3603)
GloMax® Multi+ Detection System (Cat.# E9032)

Compound Preparation and Dilutions: Terfenadine and ionomycin (10mM) were prepared in 100% DMSO. Compounds were serially diluted twofold into 100% DMSO (10mM–39µM). These dilutions were further diluted 50-fold into cell culture medium. Fifty microliters of the medium-diluted compounds were added per sample well. For CellTox™ Green no-step samples, 10µl of CellTox™ Green Dye was first diluted in 5ml of cell culture medium before dispensing compound dilutions.

Cell Preparation: HEK 293 cells were trypsinized, collected and counted. Cells were centrifuged at 300 × g for 5 minutes and resuspended to 1.0 × 105 cells/ml in DMEM + 10% FBS. Cells (50µl) were added per sample well (5,000 cells/well) containing diluted compound.

Cytotoxicity Assay: Compound dilutions and cells were mixed in replicates of six in three 96-well tissue culture plates per multiplexing experiment: white plates (for multiplexing with CellTiter-Glo® Assay), black plates (for multiplexing with CellTiter-Fluor™ Assay), or black plates with clear bottoms (for multiplexing with CellTiter 96® AQueous One Solution Assay). One plate contained CellTox™ Green Dye in medium (no step), and one plate contained medium only without CellTox™ Green Dye (no-CellTox™ Green control). Cells were incubated with compound for 6 hours at 37°C, 5% CO2. After incubation, cytotoxicity was monitored by detecting CellTox™ Green fluorescence using the blue filter set (excitation 490nm; emission 510–570nm) on the GloMax Multi+ Detection System (no-step plate).

Viability Assays

CellTiter-Glo® Luminescent Cell Viability Assay: After detecting cytotoxicity with the CellTox™ Green Assay, 100µl of CellTiter-Glo® Reagent was added to each sample well and incubated for 10 minutes at room temperature (22–25°C). Luminescence (ATP) was detected on the GloMax®-Multi+ Detection System using the luminescence module with an integration time of 0.5 seconds.

CellTiter-Fluor™ Cell Viability Assay: After measuring cytotoxicity with the CellTox™ Green Assay, 20μl of CellTiter-Fluor™ Reagent (prepared as 10μl substrate in 2ml Assay Buffer) was added to each sample well, mixed briefly by orbital shaking and incubated for 30 minutes at 37°C, 5% CO2. Fluorescence (viability) was detected on the GloMax®-Multi+ Detection System using the AFC optical kit (excitation 365nm; emission 410–460nm).

CellTiter 96® AQueous One Solution Cell Proliferation Assay: After determining cytotoxicity with the CellTox™ Green Assay, 20µl of CellTiter 96® AQueous One Solution reagent was added to each sample well and incubated for 4 hours at 37°C, 5% CO2. Absorbance at 450nm (A450, viability) was measured on the GloMax®-Multi+ Detection System at 1 and 4 hours.

Results

To determine if CellTox™ Green Dye affects the CellTiter-Glo® Viability Assay, cells were treated with two compounds and tested for viability in the presence and absence of CellTox™ Green Dye using no-step and endpoint methods. Cells were treated with serial dilutions of the compounds for 6 hours before determining cytotoxicity using the CellTox™ Green Assay followed by adding CellTiter-Glo® Reagent and determining cell viability (Figure 2, Panels A and B). The CellTox™ Green Assay showed that as compound concentration increased, there was an increase in cytotoxicity. At the highest concentration, terfenadine resulted in a 25-fold increase and ionomycin resulted in a 14-fold increase in fluorescence compared to vehicle control. Cell viability decreased as each compound increased in concentration as determined by the CellTiter-Glo® Assay. With CellTox™ Green Dye present, there was a 23% reduction in CellTiter-Glo® Assay relative light units (RLU) with cells treated with terfenadine and 20% reduction for ionomycin. This decrease in signal was proportional across compound concentrations and did not affect the IC50 calculations for each compound. Previous experiments have shown that a similar reduction in luminescence occurs if CellTox™ Green Dye is added just before reading luminescence, suggesting the CellTox™ Green Dye does not affect cell viability. The fold luminescent response was very high even with CellTox™ Green Dye present (1,000-fold for terfenadine and 4,000-fold for ionomycin). CellTox™ Green Dye was also tested as an endpoint assay, and similar results were observed (data not shown).

Figure 2. Multiplex assays with CellTiter-Glo® Viability Assay and CellTox™ Green Cytotoxicity Assay.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents luminescent signal from CellTiter-Glo® Assay in relative light units (RLU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents luminescent signal from CellTiter-Glo® Assay in relative light units (RLU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

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To determine if CellTox™ Green Dye affects the CellTiter-Fluor™ Cell Viability Assay, cells were treated with two compounds and tested for viability in the presence and absence of CellTox™ Green Dye using no-step and endpoint methods. Cells were treated with serial dilutions of the compounds for 6 hours before assessing cytotoxicity using the CellTox™ Green Assay followed by adding CellTiter-Fluor™ Reagent and determining cell viability (Figure 3, Panels A and B). The CellTox™ Green Assay showed that as the compound concentrations increased, there was an increase in cytotoxicity. Cell viability decreased as the concentration of each compound concentration increased as determined by the CellTiter-Fluor™ Cell Viability Assay. There was no significant difference in relative fluorescent units (RFU) in the absence or presence of CellTox™ Green Dye. CellTox™ Green Dye was also tested as an endpoint assay, and we observed a similar increase in cytotoxicity and similar effects on the CellTiter-Fluor™ Cell Viability Assay signal (data not shown).

Figure 3. Multiplexing the CellTiter-Fluor™ Cell Viability Assay with the CellTox™ Green Cytotoxicity Assay.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents signal from CellTox™ Green Dye and left axis represent signal from CellTiter-Fluor™ Viability Assay, both in relative fluorescent units (RFU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents signal from CellTox™ Green Dye and left axis represent signal from CellTiter-Fluor™ Viability Assay, both in relative fluorescent units (RFU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

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To determine if CellTox™ Green Dye affects the CellTiter 96® AQueous One Solution Cell Proliferation Assay, cell were treated with terfenadine and tested for viability in the presence and absence of CellTox™ Green Dye using the no-step method. Cells were treated with a serial dilution of compound for 6 hours followed by a 1- and 4-hour incubation with the CellTiter 96® AQueous One Reagent. After incubation, cytotoxicity signals were measured before assessing viability using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Figure 4, Panel A and B). The CellTox™ Green Assay showed that as the compound concentrations increased, there was an increase in cytotoxicity. At the highest concentration, terfenadine resulted in a 14-fold increase in fluorescence at 1 hour and 50-fold increase at 4 hours compared to vehicle control. Cell viability decreased as the concentration of each compound increased as determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay. With CellTox™ Green Dye present, there was a 5% increase in absorbance (A450nm) with cells treated with terfenadine at both 1 and 4 hours. This increase in signal was proportional across compound concentrations and did not affect the IC50 calculations for the compound. CellTox™ Green was also tested as an endpoint assay and similar increase in cytotoxicity and similar effects on the CellTiter 96® AQueous One signal were observed (data not shown).

Figure 4. Multiplexed assays using CellTiter 96® AQueous One Solution Cell Proliferation Assay and CellTox™ Green Cytotoxicity Assay.

HEK 293 cells were treated for 14 hours with a serial dilution of terfenadine, followed by either a 1-hour (Panel A) or 4-hour (Panel B) treatment with CellTiter 96® AQueous One Solution Reagent and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents absorbance at 450nm from the CellTiter 96® AQueous One Solution Assay. Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

HEK 293 cells were treated for 14 hours with a serial dilution of terfenadine, followed by either a 1-hour (Panel A) or 4-hour (Panel B) treatment with CellTiter 96® AQueous One Solution Reagent and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents absorbance at 450nm from the CellTiter 96® AQueous One Solution Assay. Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

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Conclusion

The CellTox™ Green Cytotoxicity Assay can be multiplexed with three viability assays, each of which uses a different detection method: CellTiter-Glo® Luminescent Viability Assay, the fluorescent CellTiter-Fluor™ Cell Viability Assay, and the absorbance-based CellTiter 96® AQueous One Solution Cell Proliferation Assay. Despite the no-step method for CellTox™ Green Dye affecting the signal in two of the three subsequent viability assays, the effect was consistent across all compound doses and did not change IC50 values. Multiplexing offers the advantage of generating data for both viability and cytotoxicity from the same sample without creating parallel plates for both measurements. The no-step addition of CellTox™ Green Dye allows easy monitoring of cytotoxicity, reducing variability and eliminating extra steps.

References

  1. Niles A et al. (2012) Measuring cytotoxicity in real time with a highly stable green dye. Promega PubHub.

How to Cite This Article

Hook B, Bratz M and Schagat T. Gain More Informative Data by Multiplexing a Fluorescent Real-Time Cytotoxicity Assay with Luminescent, Fluorescent or Colorimetric Viability Assays. [Internet] June 2013. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/multiplexing-a-fluorescent-real-time-cytotoxicity-assay-with-luminescent-fluorescent-or-colorimetric/

Hook B, Bratz M and Schagat T. Gain More Informative Data by Multiplexing a Fluorescent Real-Time Cytotoxicity Assay with Luminescent, Fluorescent or Colorimetric Viability Assays. Promega Corporation Web site. http://www.promega.com/resources/pubhub/multiplexing-a-fluorescent-real-time-cytotoxicity-assay-with-luminescent-fluorescent-or-colorimetric/ Updated June 2013. Accessed Month Day, Year.

CellTiter 96, CellTiter-Glo and GloMax are registered trademarks of Promega Corporation. CellTiter-Fluor and CellTox are trademarks of Promega Corporation.

Figures

Figure 1. CellTox™ Green Cytotoxicity Assay.

Panel A. CellTox™ Green Dye binds DNA of cells with impaired membrane integrity. Panel B. Schematic diagram of methods for delivering the CellTox™ Green Dye.

Panel A. CellTox™ Green Dye binds DNA of cells with impaired membrane integrity. Panel B. Schematic diagram of methods for delivering the CellTox™ Green Dye.

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Figure 2. Multiplex assays with CellTiter-Glo® Viability Assay and CellTox™ Green Cytotoxicity Assay.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents luminescent signal from CellTiter-Glo® Assay in relative light units (RLU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents luminescent signal from CellTiter-Glo® Assay in relative light units (RLU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

//embed.widencdn.net/img/promega/rwizcqyohe/640px/11697MA.jpeg?u=7fvzhm
Figure 3. Multiplexing the CellTiter-Fluor™ Cell Viability Assay with the CellTox™ Green Cytotoxicity Assay.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents signal from CellTox™ Green Dye and left axis represent signal from CellTiter-Fluor™ Viability Assay, both in relative fluorescent units (RFU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

HEK 293 cells were treated for 6 hours with a serial dilution of terfenadine (Panel A) or ionomycin (Panel B) and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents signal from CellTox™ Green Dye and left axis represent signal from CellTiter-Fluor™ Viability Assay, both in relative fluorescent units (RFU). Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

//embed.widencdn.net/img/promega/3uuxzvbqe1/640px/11698MA.jpeg?u=7fvzhm
Figure 4. Multiplexed assays using CellTiter 96® AQueous One Solution Cell Proliferation Assay and CellTox™ Green Cytotoxicity Assay.

HEK 293 cells were treated for 14 hours with a serial dilution of terfenadine, followed by either a 1-hour (Panel A) or 4-hour (Panel B) treatment with CellTiter 96® AQueous One Solution Reagent and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents absorbance at 450nm from the CellTiter 96® AQueous One Solution Assay. Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

HEK 293 cells were treated for 14 hours with a serial dilution of terfenadine, followed by either a 1-hour (Panel A) or 4-hour (Panel B) treatment with CellTiter 96® AQueous One Solution Reagent and tested for viability and cytotoxicity. CellTox™ Green Dye was incorporated into the medium (no-step method) for half of the samples (+ CellTox™ Green Cytotoxicity Assay). Right axis represents fluorescent signal from CellTox™ Green Dye in relative fluorescent units (RFU) and left axis represents absorbance at 450nm from the CellTiter 96® AQueous One Solution Assay. Each point and standard deviation represents six replicates. IC50 values are listed for samples with and without CellTox™ Green Dye.

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