Cellular Health and Apoptosis
Programmed cell death (PCD) is an important process for maintaining homeostasis in multicellular organisms through the removal of cells which may have been damaged or compromised by various means. When this process is de-regulated, cancer, autoimmunity and neurogenerative disorders have been shown to occur. Therefore, development of drug targets that affect these processes is an active area of drug discovery research. The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay is an in vitro assay that measures the exposure of phosphatidylserine (PS) on the outer leaflet of cell membranes during the apoptotic process. The assay is non-lytic and allow for multiple readings from a single assay well. The assay contains a Necrosis Detection Reagent which provides a real-time measurement of cells that have progressed to secondary necrosis.
The assay was developed using the GloMax® Discover System due to its luminescence sensitivity, dynamic range, cross-talk and reproducibility. The assay is performed as follows. For detailed instructions and assay notes, see the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Technical Manual #TM507.
- Plate 50µl of cells (U937 cells in this example) in growth medium (RPMI-1640 + 10% FBS) into each well of a white TC-treated 96-well plate (10,000 cells/well). Include a set of control wells with no cells present (growth medium only) to determine background luminescence and background fluorescence.
- Perform a 10-point, 3-fold serial dilution of rhTRAIL (an extrinsic apoptosis inducer) in growth medium at 4X the desired final concentration. Be sure to include a set of control wells with no rhTRAIL (untreated controls). Add 50µl of the 10-point, 3-fold serial dilution of rhTRAIL (and no compound control) to the appropriate replicate wells in the 96-well assay plate.
- Add 100µl of 2X concentrated RealTime-Glo® Annexin V Apoptosis and Necrosis Detection Reagent in growth medium to each well.
- Incubate cells in the covered 96-well assay plate at 37°C/5%CO2 in a humidified cell culture incubator.
- Measure luminescence and fluorescence (475nmEx/500–550nmEm) on the GloMax® Discover Instrument using the RealTime-Glo® pre-programmed protocol at 0, 3.5, 6.5 and 24 hours.
Figure 1. Annexin V binding to phosphatidylserine (PS:Anx) and loss of membrane integrity in real-time. U937 cells were incubated at 37°C/5%CO2 in the presence of serial dilutions of rhTRAIL and the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Reagents. Background subtracted luminescence, RLU for Annexin V binding to phosphatidylserine (Panel A) and background subtracted fluorescence, RFU for membrane integrity (Panel B) were measured at 0, 3.5, 6.5 and 24 hours on the GloMax® Discover instrument. The time-dependent increase in luminescence (due to Annexin V binding to phosphatidylserine, panel A) that occurs prior to the time-dependent increase in fluorescence (due to loss of membrane integrity, panel B) reflects apoptosis followed by secondary necrosis.
Studying apoptosis and secondary necrosis in vitro can be easily achieved using the Promega RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay and Kit and the GloMax® Discover System. The luminescent and fluorescent readouts can be measured repeatedly from the same samples over a long-period to record the kinetics of different cell death processes. The GloMax® pre-programmed instrument protocol, instrument sensitivity and dynamic range performance provide a robust system for measuring programmed cell death in real time.
Immune Checkpoint Targets
Immune checkpoint pathways are common targets for immunotherapy drug development. CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) is an immune inhibitory receptor and a homolog of the T-cell co-stimulatory receptor CD28. It is expressed on activated CD4+ and CD8+ T cells after T cell receptor (TCR) signaling. The CTLA-4 Blockade Bioassay provides a non-radioactive, plate-based, homogeneous bioluminescent method for measuring the biological activity of CTLA-4 blocking antibodies during immunotherapy drug development. The assay consists of two cell lines—a CTLA-4 Effector Cell line and an antigen presenting cell line. CTLA-4 Effector cells stably express CTLA-4 receptor and a luciferase reporter that responds to TCR and CD28 activation. The artificial antigen presenting cells (aAPC/Raji) are Raji cells stably expressing a T cell activator protein, which activates the CTLA-4 Effector cells in an antigen-independent manner, and endogenously expressing CD80 and CD86. Co-incubation of CTLA-4 Effector cells with aAPC/Raji cells blocks CD28-activated luciferase activity. Addition of an anti-CTLA-4 blocking antibody releases the inhibitory signal, restoring CD28-activated expression of luciferase activity.
Figure 2. CTLA-4 Blockade Assay. Six-hour response to ipilimumab (an anti-CTLA-4 antibody marketed as Yervoy for metastatic melanoma) using CTLA-4 Effector Cells and aAPC/Raji Cells. Four-parameter logistic curve analysis was performed with GraphPad Prism® software. EC50 was 2.27µg/ml.
The assay was developed using the GloMax® Discover System due to its luminescence sensitivity, dynamic range, cross-talk and reproducibility. The assay is performed as follows. For detailed instructions and assay notes for various assay volumes and plate format, see the CTLA-4 Blockade Bioassay Technical Manual #TM518.
- Plate 25µl of CTLA-4 Effector Cells into white 96-well plates.
- Serially dilute anti-CTLA-4 antibody and add 25µl to the assay wells already containing Effector Cells.
- Add 25µl of aAPC/Raji cells.
- Incubate for 6 hours at 37°C/5%CO2.
- Add 75µl of Bio-Glo™ Luciferase Assay Reagent and incubate for 10 minutes.
- Measure luminescence on the GloMax® Discover using the Bio-Glo™ pre-programmed assay protocol.
Figure 3. Schematic workflow for the CTLA-4 Blockade Bioassay. Plating the CTLA-4 Effector Cell line and the artificial antigen presenting Raji cells (aAPC/Raji). For detailed notes on various assay volumes and plate format, see the CTLA-4 Blockade Bioassay Technical Manual #TM518.
The CTLA-4 Blockade Bioassay was developed and optimized using the GloMax® Discover System due to the superior performance. This integrated bioassay provides confidence that even low level cellular responses can be measured successfully.
NanoBiT® Binary Technology is a complementation system based on two NanoBiT® Luciferase subunits. The large subunit (LgBiT) has enhanced structural stability while the small subunit (SmBiT) was developed for use in protein:protein interaction (PPI) assays. LgBiT and SmBiT subunits are fused to two interacting target proteins. When these proteins interact, the subunits are brought into close proximity to form a functional enzyme that generates a bright, luminescent signal.
The assay was developed using the GloMax® Discover System due to its luminescence sensitivity, dynamic range, cross-talk and reproducibility. The assay is performed as follows. For detailed instructions and assay notes, see the NanoBiT® Protein:Protein Interaction System Technical Manual #TM461.
Day 1 (Plate cells)
- Use the inner 60 wells of a 96-well plate. Plate HEK293 cells in complete medium (DMEM + 10% FBS) in 100µl/well at a concentration of 1 × 105 cells/ml.
- Incubate cells overnight at 37°C/5%CO2.
- Add 200µl DPBS to the outside wells.
Day 2 (Transfection)
- Dilute plasmid DNA encoding FRB-LgBiT Control Vector and FKBP-SmBiT Control Vector in OptiMEM® I Reduced Serum Medium (Life Technologies Cat.# 11058) to 6.25ng/µl for each construct.
- Add FuGENE® HD Transfection Reagent at a 3:1 lipid-to-DNA ratio.
- Incubate at ambient temperature for 10 minutes.
- Add 8µl of the transfection mix to each well.
- Incubate at 37°C/5% CO2for 24 hours.
Day 3 (Assay)
- Exchange medium to 100µl/well pre-warmed OptiMEM® I Reduced Serum Medium.
- Incubate at 37°C/5% CO2 for 30 minutes.
- Equilibrate Nano-Glo® LCS Dilution Buffer to room temperature.
- Make Nano-Glo® Live Cell Reagent by combining 1 volume of Nano-Glo® LCS Dilution Buffer (a 20-fold dilution), creating a 5X stock to mix with cell culture medium.
- Prepare 13.5X stocks of rapamycin for dose response by diluting compound in OptiMEM: Final concentration in nM: 100.0, 31.8, 10.0, 3.18, 1.0, 0.318, 0.010, 0.031, 0.01, vehicle control (DMSO).
- Prepare GloMax® Discover by selecting the Nano-Glo® Live Cell pre-programmed protocol. Select the inner 60 wells to be measured.
- Add 25µl of 5X Nano-Glo® Live Cell Reagent per well and place plate in GloMax® Discover instrument.
- Protocol is set as follows:
a. Measure every minute at 0.5sec/well for 30 minutes (30 reads).
b. A prompt to add compound will appear. Add 10µl of a 13.5X stock of rapamycin titration.
c. A prompt to close the door and start read will appear. The plate will be read 30 times at 1-minute intervals.
Figure 4. Rapamycin response kinetics. Using the GloMax® Discover and the Nano-Glo® Live Cell Assay program, signal was measured from cells expressing the FRB:FKBS NanoBiT® control pair for 30 minutes before a 13.5X titration of rapamycin was added and luminescence measured for an additional 30 minutes.
Figure 5. Rapamycin dose response. Panel A represents the average luminescent signal after 30 minutes rapamycin exposure. Panel B represents the fold response after 30 minutes rapamycin exposure.
Protein-protein interaction dynamics can be assessed in real-time using the NanoBiT® PPI Assay and the GloMax® Discover System. A true measure of the biological response can be ascertained due to the sensitivity of the luminescence assay and instrument system.
Nucleic Acid Quantitation
Accurate quantitation of nucleic acid is critical for many biological applications including cloning, amplification and next-generation sequencing. The QuantiFluor® dsDNA System contains a fluorescence DNA-binding dye that sensitively and specifically quantitates small amounts of double-stranded DNA (dsDNA) in solution.
Using the QuantiFluor® dsDNA system is made easy with the GloMax® Discover System due to its integrated protocol, fluorescence sensitivity and reproducibility. The assay is performed as follows. For detailed instructions and assay notes, see the QuantiFluor® dsDNA System Technical Manual #TM346.
- Warm all assay components to room temperature before use. The QuantiFluor® dsDNA Dye is dissolved in 100% DMSO and frozen at or below 40°C. Prior to dilution, thaw dye at room temperature, protected from light.
- Prepare 1X TE buffer by diluting the 20X TE Buffer 20-fold with Nuclease-Free Water.
- Dilute the QuantiFluor® dsDNA Dye with 1X TE buffer. For the standard curve, perform a 1:200 dilution. Prepare enough QuantiFluor® dsDNA Dye working solution to quantitate both standards and unknown samples.
- Quantitation of unknown samples requires comparison of the unknown samples to a standard curve of dsDNA. Generate a standard curve appropriate for the expected dsDNA concentration range of your unknown samples and your sample analysis setup. We recommend preparing a standard curve using dsDNA of a similar size as the dsDNA you wish to quantitate. For example, if you are quantitating genomic DNA, you should prepare a standard curve using a genomic DNA sample of known concentration. The Lambda DNA Standard included is 48.5kg.
- For the dsDNA standard curve (0.2–1,000ng/ml), dilute the Lambda DNA Standard 1:50 in 1X TE buffer to a concentration of 2ng/µl. For example, add 20µl of Lambda DNA Standard to 980µl of 1X TE buffer.
- Prepare the standard samples shown in Table 1 for a dsDNA standard curve.
Table 1. Preparing dsDNA Standard Curve.
||Volume of dsDNA Standard
||Volume of 1X TE Buffer (μl)
||dsDNA Amount Per 100 μl (ng)
||dsDNA Concentration Before Adding Dye (ng/ml)
||Final dsDNA Concentration After Adding Dye (ng/ml)
||250μl of Standard A
||250μl of Standard B
||250μl of Standard C
||250μl of Standard D
||250μl of Standard D
||250μl of Standard E
||250μl of Standard F
1Use 1,000μl of the 2ng/μl Lambda DNA Standard prepared in Step 1.
Figure 6. Representative dsDNA standard curve in a 96-well plate format. The final amounts of the Lambda DNA Standard in the 96-well 200µl assay format. Points represent the average of three replicates with standard deviation shown. Inset: Expanded view of the low end of the standard curve.