Using the GloMax®-Multi Detection System for Measuring Signal from Multiplexed...

Introduction

Multiplexing various cell-based assays allows the researcher to gain a more complete understanding of the biological phenomenon being studied while also saving time, reagents, cells and valuable test compounds. Promega offers a wide range of fluorescent and luminescent cell-based assays that can be successfully multiplexed^{(1) (2)} . The homogeneous format of these assays allows them to be easily combined and measured in a single multiwell plate, allowing sequential analysis of any two of the following: cell viability, cytotoxicity, apoptosis and reporter activity.

Instrument quality is paramount for accurate measurement of signal from multiplexed assays. To address this issue, Promega now offers the GloMax®-Multi Detection System Cat.# E7031, an instrument designed for optimal detection of absorbance, fluorescence and luminescence signals over a broad dynamic range. Unlike other multifunction readers, the GloMax®-Multi System contains independent modules and circuitry for detecting each signal type. This creates an instrument with enhanced sensitivity and dynamic range in all modes.

To demonstrate the versatility and sensitivity of the GloMax®-Multi Detection System, the instrument was used to detect signal output from three sets of multiplexed cell-based assays: the CellTiter-Blue® Cell Viability Assay (Cat.# G8080; fluorescent cell viability assay) and Caspase-Glo® 3/7 Assay (Cat.# G8090; luminescent apoptosis assay); the CytoTox-Fluor™ Cytotoxicity Assay (Cat.# G9260; fluorescent cytotoxicity assay) and CellTiter-Glo® Luminescent Cell Viability Assay (Cat.# G7570); and the EnduRen™ Live Cell Substrate (Cat.# E6481; luminescent reporter assay) and Apo-ONE^® Homogeneous Caspase-3/7 Assay (Cat.# G7792; fluorescent apoptosis assay).

Results

CellTiter-Blue® Cell Viability Assay Multiplexed with Caspase-Glo® 3/7 Assay

Fluorescent signal from resazurin (provided in the CellTiter-Blue® Reagent) reduction to resorufin by live cells was inversely proportional to luminescent signal generated by apoptotic cells, cleaving the proluminescent caspase-3/7 DEVD-aminoluciferin substrate (provided in the Caspase-Glo® 3/7 Reagent; Figure 1). These data demonstrate a correlation between loss of cellular reductive potential (viability) and gain of apoptotic response as a function of increasing staurosporine concentration.

Figure 1. Multiplexing the CellTiter-Blue® Cell Viability and Caspase-Glo® 3/7 Assays.

Jurkat cells (10,000 cells/well, 100µl/well) were treated with various concentrations of staurosporine for 4.5 hours in a Costar® white-wall, 96-well plate. Multiplexing was performed as described in reference 1. Twenty microliters of CellTiter-Blue® Reagent (diluted 1:4 in Dulbecco’s PBS) was then added to each well. The cells were incubated an additional 3 hours at 37°C, then cell viability was assessed on the GloMax®-Multi System using the Green (525nm_Ex/580–640nm_Em) optical kit. After the fluorescence signal was measured, 120µl of Caspase-Glo® 3/7 Reagent was added to each well. The multiwell plate was incubated for 1 hour at room temperature, and the luminescent signal measured using the GloMax®-Multi System.

CytoTox-Fluor™ Cytotoxicity Assay Multiplexed with CellTiter-Glo® Luminescent Cell Viability Assay

Fluorescent signal from Rhodamine 110, the cleaved product of the fluorogenic peptide substrate bis-alanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110; used in the CytoTox-Fluor™ Reagent to measure “dead-cell activity”), correlated with increasing concentrations of ionomycin (Figure 2). There was a corresponding decrease in cellular ATP (used as the limiting reaction component for the CellTiter-Glo® Reagent as an indicator of cell viability) as a function of ionomycin concentration. These data illustrate that as the ionomycin concentration increased, there was a corresponding increase in cytotoxicity and decrease in cell viability.

Figure 2. Multiplexing the CytoTox-Fluor™ Cytotoxicity and CellTiter-Glo® Luminescent Cell Viability Assays.

Jurkat cells (10,000 cells/well, 100µl/well) were treated with various concentrations of ionomycin for 6.5 hours in a Costar® white-wall, 96-well plate. Following the protocol for multiplexing in the CytoTox-Fluor™ Cytotoxicity Assay Technical Bulletin #TB350, 10µl of CytoTox-Fluor™ Cytotoxicity Assay Reagent was added to each well, and the cells were incubated for 1 hour at 37°C. The cell cytotoxicity was assessed on the GloMax®-Multi System using the Blue (460 nm_Ex/515–580 nm_Em) optical kit. After measuring the fluorescence signal, 110µl of CellTiter-Glo® Reagent was added to each well. The plate was incubated for 20 minutes at room temperature and luminescent signal measured using the GloMax®-Multi System.

EnduRen™ Live Cell Substrate Multiplexed with Apo-ONE® Homogeneous Caspase-3/7 Assay

In the presence of the EnduRen™ Live Cell Substrate, cells constitutively expressing Renilla luciferase generated luminescence. This was used as a measure of protein expression and cell health. Renilla expression decreased proportionally with increasing concentrations of staurosporine (Figure 3). There was also a proportional increase in the cleavage of the profluorescent caspase-3/7 consensus substrate, rhodamine 110 bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-aspartic acid amide; Z-DEVD-R110), indicating cell apoptotic response to staurosporine. These data demonstrated the loss of reporter protein expression and activation of the apoptotic response with respect to increasing staurosporine concentration.

Figure 3. Multiplexing the EnduRen™ Live Cell Substrate and Apo-ONE® Homogeneous Caspase-3/7 Assays.

HEK293 cells stably expressing Renilla luciferase from a CMV promoter were cultured at 10,000 cells/well for 24 hours. The cells were then treated with various concentrations of staurosporine for 16 hours in a Costar® white-wall, 96-well plate (100µl/well). Multiplexing was performed as described in reference 1. Ten microliters of 60µM EnduRen™ Substrate was added to each well and incubated for 2 hours at 37°C (per the EnduRen™ Live Cell Substrate Technical Manual #TM244). Luminescent reporter activity was measured on the GloMax®-Multi System, then 100µl of Apo-ONE® Reagent was added to each well. The plate was incubated for 1 hour at room temperature, and apoptotic activity was assessed on the GloMax®-Multi System using the Blue (460nm_Ex/515–580nm_Em) optical kit.

Conclusion

The GloMax®-Multi Detection System is a sensitive and cost-effective “all-in-one” instrument ideally suited for the researcher who wants to use a wide assortment of assay types and perform assay multiplexing. The GloMax®-Multi System effectively detects signal from fluorescent, luminescent and colorimetric assays for cell viability, cytotoxicity, apoptosis and reporter activity with little background noise. This is especially important when measuring bioluminescence, since excessive background noise will limit dynamic range. To learn more about the GloMax^®-Multi Detection System, please visit the GloMax®-Multi System catalog entry.