Multiplexing Cell-Based Assays:Get More Biologically Relevant DataFall 2010
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Multiplexing assays for more informative data
• Plate-based assays for viability, cytotoxicity and apoptosis measurement
• Using multiplex assays to understand cell death mechanism
• Monitoring cell response in multiple applications
Distinct enzyme chemistries allow multiplexing in the Dual-Luciferase® Assay
Mix 20µl of cell lysateand 100µl of Luciferase Assay Reagent II in the tube.
Measure the light produced by firefly luciferase.
Add 100µl of Stop & Glo Reagent to the tube.
Measure the light produced byRenilla Luciferase.
Renilla Luciferaselevels vary little with treatment
Control Renilla corrects for:• cell death• bad transfection
The Stop & Glo® Reagents
quench firefly luminescence
and provide the substrate for
Renilla luciferase
khooper
Sticky Note
Many of you are multiplexing if you’ve ever used the Dual-Luciferase Assay System. This assay is possible because we use two different luciferases with distinct chemistries that allow us to measure one then the other. The Renilla control revolutionized reporter assays because it corrected for cell death during treatment and could even save you if you had a bad transfection.
Understand overall cellular response to treatment
LIVE-CELLPROTEASE
GF-AFCAFC
If only reporter activity is measured, the impact of
compound treatment on cell health may be overlooked.
khooper
Sticky Note
Many of you, still continue to use single reporter assays. Maybe you’ve had problems with reporter cross talk or you have stably transfected cell line or any number of reasons. Let’s look at this example at the bottom. You see a rise then a fall in reporter activity (green line) with increasing ionomycin. You might think that the reporter is being upregulated then downregulated. However, if you’d just added one simple assay upstream, you’d see that you really weren’t downregulating the promoter activity but killing the cells. Its important to consider cell health during any cell-based assay.
Measure live/dead cells upstream of any of our bioluminescent assay is straightforward and rather easy. Simply treat your cells like normal, then simply add the CellTiter-Fluor, CytoTox-Fluor or MultiTox-Fluor reagent to the cells and incubate for 30min at 37°C. Pop it into a fluorometer and measure the assay or assays. Your next step is to perform the bioluminescent assay using the normal protocol. There is one simple thing you change from the standard protocols for these three assays: you change the concentration of the reagent so that the fluorescent assay and the bioluminescent assay can fit in the well. All three fluorescent kit protocols include instructions for making a concentrated reagent for multiplexing.
Normalize Reporter Data to viable cells
Steady-Glo® Luciferase Assay
Bright-Glo™ Luciferase Assay GloResponse™ NF-kB Cells at
5K to 25K cells/well
Raw Luminescence
Raw Luminescence
Normalized
Normalized
khooper
Sticky Note
Here’s an artificial example of how adding CellTiter-Fluor can help you. We purposely pipetted 5000 to 25000 GloResponse cells into the wells of plate. We then performed the CellTiter-Fluor assay followed by either a Bright-Glo Luciferase or Steady-Glo Luciferase Assay. As you can see, the raw luminescent signal is all over the place. However if you normalize it to the AFC fluorescence, the noise calms down and you get something you can work with. Here’s another example...
Monitor changes in GSH levels in cells under stress
UltraGlo™
Luciferase+ ATP
Light
A change in GSH levels is important in assessment of toxicological responses and is an indicator of oxidative stress, potentially leading to apoptosis or cell death.
khooper
Sticky Note
Like I said earlier, apoptosis is a controlled event and typically results from a cellular stress that starts the cascade of events. One stressor is the loss of glutathione in the cell. Glutathione can be lost through detoxifying a drug or through neutralization of oxidative stress. We devised a luminescent assays to measure the amount of oxidized glutathione or GSH in cells. We use glutathione-S-transferase to transfer a side group from a proluciferin to GSH which generates luciferin. The luciferin is converted to light in a luciferase reaction and the amount of light produced is proportional to the amount of GSH in the cells. This graph simply shows that GSH levels are proportional to cell number in healthy cells.
Multiplexing Viability and GSH Levels
Add MultiTox-Fluor Reagent
Add GSH-Glo Reagent,30min
Incubate 30 min
Record Fluorescence
Record Luminescence
Remove medium(GSH background)
Add Luciferin Detection Reagent, 15min
GSH Synthesis Inhibitor
Again, timing is everything.In this window, there is no change in viability but be assured, a lack of GSH will lead to cytotoxicity.
khooper
Sticky Note
Here’s the full workflow for this assay. The cells are treated with BSO (too hard to say the real name) then MultiTox-Fluor reagent is added and 30 minutes later, viable and dead cells are measured. The process continues with the normal GSH-Glo Glutathione Assay. The media is removed from the cells (basically to lower background) and the GSH-Glo Reagent containing the GST and proluciferin is added. After 30 minutes, the luciferin detection reagents are added and the luminescence recorded. This was a 24 hour incubation. Again, timing is everything. In this assay timeframe, BSO is not toxic but, rest assured, cells need GSH and lack of it will cause cytotoxicity.
Histone Deacetylases are cancer targets
• Acetylated histones have tight structure, restricting transcription
• Deacetylated histones have a loose structure, encouraging transcription
Hess-Stumpp et. al. 2007
• Balance of acetylation and deacetylation is important & an imbalance is not good.
HDAC-Glo™ I/IISubstrate
HDAC DeveloperReagent
UltraGlo™Luciferase
ATPLight
khooper
Sticky Note
Here’s another example with a new bioluminescent kit that should be available this year called HDAC-Glo™ I/II Assay. Histone Deacetylases are major cancer targets. Inhibition of their activity promotes many pathways promoting cancer cell death. The acetylation state of histones is important. Acetylated histones have a tight structure and restriction transcription. Deacetylated histones are more open and encourage transcription. The balance is important for a healthy cell. When things get out of whack, bad things like cancer can occur. HDAC-Glo is design to assess the general level of class I or class II histone deacetylases. The assay uses a peptide containing an acetylated lysine side change. Removal of the acetyl group allows a protease in our developer reagent to cleave the peptide forming aminoluciferin. The light produced in the reaction is proportional to HDAC I/II activity.
Help identifying on- and off-target effects
iCell™ U937
Multiplexed measurement of HDAC inhibitor activity with HDAC-Glo™ I/II and cytotoxicity with Multitox-Fluor TM Reagent clearly delineates cell lines that are sensitive to HDAC inhibitors. Exposure of two cell lines to apicidin for 24h followed measurement identified the HDAC inhibitor sensitive cell line and showed good correlation between HDAC inhibition and cytotoxicity.
No apparent cytotoxicity Cytotoxicity
khooper
Sticky Note
This, too, can be multiplexed and assayed much like what I showed you earlier with ApoTox-Glo. You can multiplex viability and cytotoxicity with the HDAC inhibition to see if a drug has a target specific effect. In this case, it obviously does. The U937, a human leukemic monocyte cell line, responds to HDAC inhibition with cytotoxicity. iCells appear to be fine.
