Multiplexing fluorogenic esterase-based viability assay with luciferase assays

Graphical abstract


Method details
Cell-based reporter assays are widely used in drug discovery programs, and luciferase is one of the most widely used reporter enzymes due to its high sensitivity and wide dynamic range. In cell-based high-throughput screening, cytotoxic compounds are a major source of false positives, so multiplex measurements of cell viability and luciferase activity can improve the accuracy of hit selection and reduce the time and cost of secondary, confirmatory assays.
In luciferase-based reporter assays involving transient transfection, normalization of the luciferase activity is essential to account for well-to-well variations of transfection efficiency and cell number. For this purpose, an additional reporter plasmid encoding an orthogonal enzyme, such as betagalactosidase [1] or Renilla luciferase [2,3] is co-transfected, and its enzymatic activity is used for normalization. In some cases, however, monitoring processes of interest requires the use of a stable cell line expressing the luciferase-based reporter. For example, assays designed to monitor the stability of a protein of interest by fusing a luciferase to it often require the establishment of stable cell lines to obtain a reproducible response [4]. Then, the requirement of a stable cell line makes the cotransfecting strategy impractical. For normalization purposes, cell viability assays are sometimes performed in parallel, or total protein concentration is occasionally determined from aliquots of the lysate [5]. However, such protocols require additional resources or multiple steps for protein assay, and are not practically suitable for high-throughput assays on 96-or 384-well plates.
Several protocols, which combine resazurin-based cell viability assay or protease-based viability assay with luciferase assay, have been developed to enable multiplex determination of cell viability (number) and luciferase activity [6][7][8]. Although these protocols work well for assays monitoring relatively slow processes, such as changes of mRNA stability or transcriptional activity, they are not suitable for monitoring faster processes, including protein degradation, due to the relatively slow conversion of resazurin to fluorescent resorufin (usually >1 h) or Gly-Phe-AFC (7-amino-4trifluoromethylcoumarin) protease substrate (>30 min) within live cells. Generally, transcriptional assay requires 12-24 h for treatment of cells with test compounds, while small molecule-mediated protein degradation is detectable within several hours and in some cases, in less than one hour [4,[9][10][11][12][13]. Thus, for our on-going research on small molecule-mediated protein degradation, we required a multiplex assay that monitors both cell viability (number) and luciferase assay within a short time, preferably after cell lysis to stop cellular processes, including degradation pathways, as this allows examination of degradation processes on a shorter time scale.
Here, we describe a multiplex luciferase assay protocol that allows sequential measurement of endogenous esterase activity as a measure of cell number and luciferase activity in cell lysate (Fig. 1A). Among available esterase substrates tested, we found that a fluorogenic esterase substrate CytoRed [14] was compatible with luciferase activity measurement ( Fig. 1B-D). Basic characterization of the protocol was performed with cells stably expressing HaloTag protein fused with emerald luciferase (ELuc), a firefly-type luciferase derived from Brazilian click beetle (Fig. 1E) [15][16][17], and estrogen receptor alpha (ER) fused with ELuc (ER-ELuc) [4].

Procedure for CytoRed-luciferase dual assay
The following is a representative procedure for a 96-well plate assay. Fig. 1A illustrates how this multiplex assay works.
1 Seed cell lines expressing a luciferase construct at a density appropriate for the luciferase assay, in a white 96-well clear-bottomed plate (100 mL/well), and incubate in a humidified CO 2 incubator for an appropriate time. 2 If required, treat cells with test compounds for an appropriate time.
3 Prepare 20 mM CytoRed working solution and luciferin working solution as described in the recipes section. The working solutions are stable at least for an hour when protected from light. 4 Remove medium with an aspirator equipped with a multichannel adaptor. Proceed to the next step within a few minutes to avoid drying of the cells.
5 Add 50 mL/well of the CytoRed working solution to the plate with a multichannel pipette and a reagent reservoir, and immediately measure fluorescence on an appropriate microplate reader (Ex. 570 nm/Em. 615 nm, bottom read mode, EnVision plate reader from PerkinElmer). 6 Incubate the plate at room temperature or 37 C for 10 min, and measure the fluorescence again. For analysis, subtract the initial fluorescence from the fluorescence after incubation. Alternatively, the plate reader's kinetic measurement mode can also be used for automatic measurement. Note that longer incubation at this step results in weaker luminescence in the luciferase assay.
7 Add 50 mL/well of the luciferin working solution to the plate, and measure the luminescence after 1 min on an appropriate plate reader (we used an EnVision plate reader in ultra-sensitive luminescence mode with 10 msec exposure). Under the assay conditions (glow-type luciferase assay), luminescence lasts for more than 10 min, but gradually decreases in intensity.

Method validation
First, we examined the impact of CytoRed concentration on the fluorescence intensity and the luminescence from luciferase. Cells stably expressing HaloTag-ELuc were first assayed for endogenous esterase activity with varying concentrations of CytoRed in a Triton X-100-based lysis buffer. After 10 min at room temperature, fluorescence was measured, and the luciferase activity was then assayed as described above. As shown in Fig. 2A, fluorescence from the hydrolyzed CytoRed steadily increased with increasing CytoRed concentration, and the fluorescence intensities from wells with or without cells were well separated at more than 3 mM CytoRed. The Z' factor (0.5-0.87) indicated that the assay conditions were suitable for screening purposes [18]. CytoRed concentrations of less than 30 mM did not interfere with luciferase activity in luciferase assay of the same sample. Thus, we selected 20 mM CytoRed as an optimal condition for the multiplex assay. We also tested p-nitrophenyl acetate and pnitrophenyl isobutyl carbonate as colorimetric esterase substrates, but a sufficient signal could not be achieved even with a longer incubation time (up to 1 h) or a higher concentration (1 mM) of the substrate, at which significant interference with the luciferase assay was observed. Note that a longer incubation period at this step diminishes luciferase activity even in the absence of CytoRed. So, we recommend keeping the incubation time as short as possible (10 min). Next, we examined the dynamic range of the CytoRed esterase assay and the subsequent luciferase assay, by using serially diluted cells expressing HaloTag-ELuc. As shown in Fig. 3A, CytoRed fluorescence increased with increasing cell density, and the increase was sufficiently linear for viability (cell number) assay within the range of 3000 cells/well to 100,000 cells/well, which corresponds to 3-100% confluency. The luciferase signal, which was measured after CytoRed assay, gave excellent linearity over more than 2 orders of magnitude of cell density (Fig. 3B). Additionally, no difference in luminescence intensity was observed in the presence or absence of CytoRed, thus supporting the compatibility of CytoRed assay with luciferase assay.
Finally, we demonstrate the utility of this protocol by showing its suitability for dose-response analysis and kinetic analysis of ER degradation induced by fulvestrant, a representative selective estrogen receptor degrader (SERD) [9]. As shown in Fig. 4A, the down-regulatory effect of fulvestrant on ER-ELuc could be reproduced with this protocol, which gave an IC 50 value of 1.4 nM, in good agreement with our previously determined value (1.4 nM) [4]. Also, bortezomib, a proteasome  inhibitor, increased the ER-ELuc level in a dose-dependent manner with its EC 50 value being 55 nM, consistent with the proteasomal degradation of ER proteins. Furthermore, the kinetics of the degradation could be examined with this endpoint assay (Fig. 4B), and the half-life of the ER-ELuc in the presence of 10 mM fulvestrant was estimated to be around 30 min. Notably, no degradation was detected at less than 10 min treatment, implying the presence of a short induction period or delay that may reflect the time required for processes such as distribution of the compound into cells, binding to the receptor, and steps required for proteasomal degradation.
In summary, we have developed and validated a multiplex assay that sequentially monitors endogenous esterase activity and luciferase activity. This endpoint multiplex assay protocol takes only an additional 15 min compared to conventional luciferase assay, and allows monitoring of fast processes, providing an alternative to luciferase assay in live cells [7], which results in a significantly weaker luminescence signal. Thus, the CytoRed-luciferase multiplex assay described here is expected to be especially useful for researchers working on stable cell lines expressing luciferase constructs.

Additional information
The indicated order of measurement of esterase activity with CytoRed fluorescence and then luciferase activity is essential for success. Our attempts to measure esterase activity after luciferase activity measurement failed, possibly due to inactivation of esterase during luciferase activity measurement.