DRAFT: Rev. D A-229A APPLICATION INFORMATION Cell-Based Assays AUTOMATED MULTIPLEXING OF PROMEGA S * CELL-BASED ASSAYS WITH THE BECKMAN COULTER BIOMEK FX AND DTX 88 MULTIMODE DETECTOR Tracy Worzella and Brad Larson, Promega Corporation, Madison, WI.; Dave Daniels, Beckman Coulter, Inc., Fullerton, CA. Introduction Today s high-throughput screening facilities face increasing demands to generate more information from their existing compound libraries. This can be accomplished by two separate means. The first is to run additional assays in a singleplex format. While this may lead to the desired data, it also drives up drug development time as more assays need to be set up and run by online screening groups. Furthermore, this format drives up costs due to increased compound, labware, and tissue culture component usage. A more appealing choice is to run these same assays in a multiplexed format. This configuration helps to speed up the discovery process as more than one piece of information can be discerned with each screening run. A multiplexed format also decreases costs and variability, as multiple assays can be performed using the same set of compounds. While performing assays in a multiplexed format may indeed be more appealing to high-throughput screening facilities, there are still barriers that prevent laboratories from changing to this configuration. Short deadlines and increased pressure to identify lead compounds have created a situation where many groups do not have the time or personnel available to do new assay development. Without supporting data to show that unique assay formats will work in their lab, researchers will continue to use existing designs, even if they may not be the most advantageous for their work. What we have endeavored to do here is to provide proof of principle data, showing that multiplexed assay configurations previously tested in a manual format will also be successful in an automated high-throughput or ultra-high-throughput setting. We multiplexed our fluorescent cell viability assay, CellTiter Blue *, with our fluorescent and luminescent apoptosis assays, Apo-ONE * and Caspase-Glo * 3/7. Experiments were run using both suspension and adherent cell lines. We also combined our two live cell reporter substrates, EnduRen * and ViviRen *, with our luminescent cell viability assay, CellTiter-Glo *. These final two multiplexes were tested using agonist and antagonist models. All chemistry combinations were tested in 96-, and 384-well formats. By providing this data to facilities, researchers gain confidence that the biology and automation of multiplexed cell-based assays will work in their lab. Automated Liquid Handling Multiplexed assays were tested robotically in 96- and 384-well formats using the Beckman Coulter Biomek FX (Table ). The Biomek FX that was used for these experiments has a 96-channel head with an integrated plate gripper. An orbital plate shaker was also incorporated onto the system. This automated platform represents a system commonly found in screening facilities running in 96- and 384-well formats.
Table. Automation Platforms and Detection Instruments Used During Robotic Multiplexed Cell-based Assay Project. Company Well Format Liquid Handler Detection Instrument Beckman Coulter, Inc. 96/384 Biomek FX DTX 88 High-Throughput Detection Systems The Beckman Coulter DTX 88 was used to analyze assay plates run on the Biomek FX (Table ). The DTX 88 is a sensitive and flexible multimode detector which is configured for a wide range of applications including Glow and Dual Color Luminescence, Fluorescence Intensity (top & bottom reads), Time-resolve Fluorescence, Fluorescence Polarization, and Absorbance (UV/Vis). This reader is capable of reading 6- to 384-well plates, and can be easily integrated onto the Biomek FX liquid handler. Data and Results Multiplexed Cell Viability and Apoptosis Assays Promega s CellTiter-Blue cell viability assay was multiplexed with either Apo-ONE or Caspase-Glo 3/7. The assays were run using either suspension (Jurkat) or adherent (d293) cells. Jurkat and d293 cells were plated in the formats and concentrations stated in Table 2. Jurkat cells were then treated with a titration of Anti-FAS mab from -5 ng/ml for a period of 5 hours at 37 C/5% CO 2 to induce apoptosis (Data Not Shown). This was also accomplished in d293 cells by treating with varying doses of staurosporine ranging from -5 µm for 6 hours at 37 C/5% CO 2. CellTiter-Blue reagent was added robotically to all test plates prior to the final two hours of incubation. Fluorescence units were recorded and either Apo-ONE or Caspase-Glo 3/7 reagent were then dispensed using the various liquid handlers. The plates were incubated at room temperature for one hour, and then fluorescence or luminescence values were read. Results show the ability to robotically assay the same experimental wells with a fluorescent viability assay and either a fluorescent or luminescent apoptosis assay in 96- and 384-well plate formats (Figure ). Table 2. Cell Concentration Dispensed Per Well and Total Assay Volume for Experiments Performed in 96-, and 384-well Formats. Cell Line Well Format Cells/ Well Total Assay Volume (Cells/Trtmt/Rgnt) Jurkat 96 5. 2 µl 384 2.5 5 µl d293/hek293 96 2. 2 µl 384 5. 5 µl 2
244 293 CellTiter Blue_Apo-ONE Multiplex O/N drug, 2hr CTB, hr Apo FX/DTX 88 RFU (viability) 8 4 6 2 2 3 4 5 Staur [µm] 4 35 3 25 2 5 5 RFU (apoptosis) CTB Apo-ONE 244 293 384 well CellTiter Blue_Caspase Glo 3/7 Multiplex O/N drug, 2hr CTB, hr CG FX/DTX 88 RFU (viability) 6 25 9 55 27 225 8 35 9 45 RLU (apoptosis) 2 2 3 4 5 Staur [µm] CTB CG 3/7 Figure. Graphs showing the coupled increase in caspase activity and decrease in cell viability measured within the same well. d293 cells were plated in 96- and 384-well format and allowed to attach for hours at 37 C/5% CO 2. Apoptosis induced by treating with varying doses of staurosporine for 6 hours at 37 C/5% CO 2. CellTiter-Blue reagent added to plates using the Biomek FX, prior to the final 2 hours of incubation. Fluorescence units were recorded by the DTX 88, and Apo-ONE or Caspase-Glo 3/7 reagent robotically added using the same liquid handler. The plates were incubated at room temperature for one hour, and fluorescence or luminescence was recorded once again by the DTX 88. Live Cell Reporter Assay with Cell Viability Induction HEK 293 cells, stably transfected with an inducible CRE/CL-pEST Renilla luciferase construct, were once again plated in the plate formats and concentrations listed in Table 2. Test plates using EnduRen Live Cell Substrate to measure reporter activity had the EnduRen reagent mixed with the cells and media at the time of plating to a final concentration of 6 µm. The following day, the CRE response element was induced using a concentration of 6 µm of isoproterenol. Plates were then incubated at 37 C/5% CO 2. Reporter response and viability was then assessed at,, 3, and 5 hours, post induction. ViviRen Live Cell Substrate was added robotically at a final concentration of 6 µm to those plates using this reagent to measure reporter activity. Renilla luminescence in both EnduRen and ViviRen plates was recorded, followed by automated addition of CellTiter-Glo reagent. Luminescence was recorded a second time to measure ATP content and cell number. 3
254 pgl4-cre EnduRen_CTG 96 Well Multiplex 6 µm Isoproterenol Treatm ent Biomek FX/DTX 88 Reader Lum. (Renilla Reporter) 75 5 25 2 3 4 5 RLU (Viability) EnduRen Tim e (Hours) CTG 2234 pgl4-cre Viviren_CTG 96 Well Multiplex 6 µm Isoproterenol Treatm ent Biomek FX/DTX 88 Reader Lum. (Renilla Reporter) 75 5 25 2 3 4 5 Tim e (Hours) RLU (Viability) ViviRen CTG Figure 2. Graphs showing detection of the CRE response element with either EnduRen or ViviRen substrate over time, while subsequently monitoring cell viability within the same well. HEK 293 cells stably transfected with an inducible CRE/CL-pEST Renilla luciferase construct were plated in 96-, and 384-well formats and allowed to attach for hours at 37 C/5% CO 2. EnduRen substrate was added to the appropriate test plates at the time of plating using the Biomek FX. Isoproterenol, at a concentration of 6 µm was added to all test plates. ViviRen substrate then robotically added to the appropriate test plates. Induction monitored at,, 3, and 5-hour time points in 96-, and 384-well formats. At each time point Renilla luminescence was recorded by the DTX 88, followed by robotic addition of CellTiter-Glo reagent. Luminescence was recorded a second time to measure ATP content and cell number. Results show the ability to track the response of a reporter gene over time, by measuring luminescent Renilla reporter activity, and subsequent luminescence for cell viability in the same experimental well (Figure 2). For this application, the Renilla reporter expression was optimal at 3 hours of treatment with 6 µm Isoproterenol in 96- and 384-well formats. The results also show the incremental response generated by the ViviRen Live Cell Substrate over the EnduRen substrate. This can be of great benefit when volume, as well as signal, is decreased in a miniaturized 384-well assay. 4
224 SV4 vs SV4/CP Enduren_CTG 96 Well Multiplex (62.5 µg/ml cyclohex) % Enduren Data Biomek FX/DTX88 SV4 CTG RLU 8 6 4 2 SV4/CP CTG SV4 Enduren SV4/CP Enduren 2 3 4 Time (Hours) 224 SV4 vs SV4/CP Enduren_CTG 384 Well Multiplex (62.5 µg/ml cyclohex) % Enduren Data Biomek FX/DTX88 SV4 CTG RLU 8 6 4 2 2 3 4 Time (Hours) SV4/CP CTG SV4 Enduren SV4/CP Enduren Figure 3. Graphs showing down regulation of pgl4[hrluc/sv4] vector, and pgl4[hrluc/sv4 CP] rapid response vector while monitoring cell viability in the same well. D293 cells bulk transfected in T-75 tissue culture flasks, with one of the two pgl4 vectors mentioned. 24 hours post-transfection, cells plated in 96-, and 384-well formats (Table ). EnduRen substrate added to appropriate test plates at the time of plating using the Biomek FX. 8 hours after plating, Cycloheximide, -5 µg/ml, robotically added to the plates. Reporter response monitored at,, 2, and 4-hour time points. Renilla luminescence recorded by the DTX 88, followed by robotic addition of CellTiter-Glo reagent. Luminescence was recorded a second time to measure ATP content and cell number. Live Cell Reporter Assay with Cell Viability Down Regulation D293 cells were bulk diluted to 2. x 4 cells/ml in T-75 tissue culture flasks and allowed to adhere overnight. The cells were then bulk transfected with either the pgl4.73[hrluc/sv4] Vector, or pgl4 [hrluc/sv4]cl-pest rapid response vector. At 24 hours post-transfection the cells were plated in the formats and concentrations described in Table 2, and allowed to attach overnight. EnduRen reagent was once again added to the media and cells during plating at a final concentration of 6 µm. The following day the cells were then treated with Cycloheximide at final concentrations of -5 µg/ml, via robotic addition. Reporter response and cell viability was measured at,, 2, and 4 hours after treatment. Renilla luminescence was recorded, followed by automated addition of CellTiter-Glo reagent. Luminescence was recorded a second time to once again measure ATP content and cell number. 5
Results show the ability to robotically measure down regulation of a reporter gene, by monitoring luminescent Renilla reporter activity, and subsequent luminescence for cell viability in the same experimental well (Figure 3). The data also demonstrates the faster response that the protein degraded CL-pEST rapid response vector has to a cellular stimulus. Summary Each of the experiments demonstrated here shows the ability of Promega s multiplexed cell-based assays to be combined with the automation and detection capabilities of the Biomek FX and DTX 88 from Beckman Coulter, Inc. The data generated robotically in 96- and 384-well volumes agree with what has been previously generated in manual formats. The single reagent additions, extended half-lives, and extreme sensitivity of each of these multiplexed assays, combined with the flexible automation and detection systems from Beckman Coulter, Inc., makes these combinations ideal for high-throughput and ultra-high-throughput laboratory settings. * Promega, CellTiter Blue, Apo-ONE, and CellTiter-Glo are registered trademarks owned by the Promega Corporation. Caspase-Glo, EnduRen, and ViviRen are all trademarks owned by the Promega Corporation. All other trademarks are the property of their respective owners. B25-6837 25 Beckman Coulter, Inc.