Using Spark® Multimode Reader for Kinetic Monitoring of Cytotoxicity, Viability, and Apoptosis

It can be extremely challenging to assay cells in an appropriate window of time following a compound-mediated cytotoxic event. If this opportunity is missed, it could mean the difference between defining an effect as necrotic or apoptotic and can influence interpretation of the mode of action of a compound.

Monitoring cell viability and membrane integrity changes can highlight an exact point in time when primary or secondary necrotic events take place. Additional multiplexing with a caspase-3/7 assay can make it possible to distinguish membrane breakdown caused by apoptosis and secondary necrosis evolving from a cytotoxic event.

This article describes a method for kinetic monitoring of viability and cytotoxicity, using three Promega assays for assessment of cytotoxicity, viability, and apoptosis and Tecan’s Spark® multimode reader fitted with an improved fluorescence module platform.

Additional studies were carried out to assess the instrument’s ability to automatically inject caspase-3/7 reagent by employing a cytotoxicity threshold set in the reader software. Additionally, the effects of six kinase inhibitors and other compounds on K562 cell health were assessed over time.

Materials and methods

Drug compounds and cells

A small panel of drugs, representing known apoptosis-inducing kinase inhibitors and cytotoxic agents, was assembled. Staurosporine (LC Laboratories #S9300), ionomycin (Sigma #I0634), dasatinib (LC Laboratories #D3307), bosutinib (LC Laboratories #B1788), ponatinib (LC Laboratories #P7022), and imatinib (LC Laboratories #I5577) were prepared as 10 mM DMSO stocks. K562 cells are a human model of Chronic Myelogenous Leukemia with aberrant bcr/abl kinase activity; it was propagated in RPMI 1640 + 10% FBS (cell medium).

Assays

Table 1 lists the three Promega assays that were used in the experiments described here.

Table 1. Assays used for viability, cytotoxicity and apoptosis detection. RealTime-Glo and CellTox Green Reagents were added at the time of cell plating. Caspase-Glo® 3/7 reagent was injected with the Spark®

RealTime-Glo MT Cell Viability Assay

™RealTime-Glo™ MT Cell Viability Assay

  • Non-lytic, kinetic
  • Viable cells reduce pro-NanoLuc® substrate
  • NanoLuc® luciferase uses NanoLuc® substrate to produce light
  • Luminescence is proportional to the number of viable cells

CellTox Green Cytotoxicity Assay

CellTox™ Green Cytotoxicity Assay

  • Non-lytic, kinetic
  • Asymmetric cyanine dye binds DNA released from dead cells
  • Dye becomes fluorescent when bound to DNA
  • Fluorescence is proportional to the number of dead cells

Caspase-Glo® 3/7 Assay

Caspase-Glo® 3/7 Assay

  • Lytic, endpoint
  • Caspase-3/7 cleaves proluminescent DEVD-luciferin substrate
  • Firefly luciferase employs luciferin substrate to generate light
  • Luminescence is proportional to caspase-3/7 activity (apoptosis)

Plate setup

K562 cells were pelleted and adjusted to 5x104 cells/mL in cell medium. CellTox Green (Promega #G8731) and RealTime-Glo (Promega #G9713) reagents were added at 2X concentration to the cell suspension.

A white 384 well plate with wells having a clear bottom (Corning #3707) was used into which 20 μl of cell suspension was filled in. Wells containing only 2X CellTox Green and 2X RealTime-Glo in medium were included as assay background controls.

Compounds were serially titrated 1:5 in cell culture medium containing 0.2% DMSO, after diluting them to 2X starting concentration in cell medium.

This is followed by adding 20 μl of 2X compound titration to the plated cells. Compound control wells containing 0.2% DMSO, 2X CellTox Green, and 2X RealTime-Glo were not included.

The final assembled assays included: 1X compound in 0.1% DMSO, 1,000 K562 cells, 1X RealTime-Glo and CellTox Green reagents, a no-cell control with 1X reagents, and 0.1% DMSO vehicle control.

Before the experiments were performed, the Spark® multimode reader was pre-equilibrated to 37 °C and 5% CO2. The moat region was filled with deionized water to prepare the small humidity cassette. The assay plate, excluding lid, was placed into the humidity cassette. The covered cassette with plate was then placed into the equilibrated instrument.

The Spark® multimode reader platform

The multiple features in the Spark® reader are beneficial for cell-based experimentation. The assay requirements for the experiments described in this article are highlighted and the corresponding Spark® feature employed during each step of the process is shown in Table 2.

Table 2. Benefits of the Spark® multimode reader for cell-based applications

Assay Requirement

Spark® Feature Used

Incubate 384 well assay plate

Temperature control (37 °C)

Maintain 5% CO2 and ambient O2

Gas control

Prevent medium evaporation

Humidity cassette with liquid reservoir

Quantify cell viability

Lid lifter, top-read luminescence, 500 ms

integration time

Quantify cytotoxicity

Monochromator, bottom-read fluorescence

(EX 485/20, EM 520/20)

Add caspase-3/7 reagent

Lid lifter, reagent injector

Quantify apoptosis

Lid lifter, top-read luminescence, 500 ms integration time

A simple workflow helped achieve a walk-away data generation. Cytotoxicity and viability were monitored in real-time. The user can add Caspase-Glo® 3/7 (Promega #G8091) reagent at specific intervals, or if specific experimental conditions were satisfied.

Plate setup for walkaway data generation

Figure 1. Plate setup for walkaway data generation

Experiment 1. Assessing apoptosis at defined treatment interval time course

First, an experiment was carried out to assess the dose- and time-dependent influences on K562 cell health after treatment with the panel of test compounds. The aim was to evaluate whether changes in cytotoxicity were any indication of cells undergoing apoptosis. The RealTime-Glo assay was used to evaluate anti-proliferative effects caused by compound treatment.

A serial titration of test compound, beginning at 100 μM concentration, was applied to the cells. For each compound, replicate titrations were set up on the plate to enable the time course study. Cytotoxicity and viability were monitored on an hourly basis. Then, a subset of the wells were injected with 40 μl of Caspase-Glo® 3/7 reagent at time 0 and predetermined time points every 8-12 hours out to 72 hours.

All wells were injected with Caspase-3/7 reagent in the dose series at each time point; the plate was incubated for a period of 30 minutes, and luminescence from the apoptosis assay was measured. At an interval of 24 hours, the stock of Caspase-Glo® 3/7 Reagent supplied by the injectors was refreshed on the instrument.

Results from Experiment 1

A variety of results were revealed from the real-time detection of cell viability and cytotoxicity changes based on length of exposure and compound concentration. In some cases, particularly at high concentration, immediate cytotoxicity was detected but at lower concentrations, cytotoxic and anti-proliferative effects emerged with time.

Examples of data obtained from ponatinib treatment of K562 cells are shown in Figure 2A and 2B. The corresponding endpoint apoptosis assay results across the dose range at the various time points in the time course are shown in Figure 2C.

While at high concentration of ponatinib (100 μM), immediate cytotoxicity with moderate caspase induction was noted, at mid-range concentration of Ponatinib (20 μM), anti-proliferative effects with moderate caspase induction were noted.

Longer term exposure to lower concentrations (≤ 4 μM) beyond 40 hours revealed the most potent caspase-3/7 induction increases in cytotoxicity and decreases in cell viability beginning to emerge.

Time and dose-dependent effects on K562 cell health following ponatinib treatment.

Time and dose-dependent effects on K562 cell health following ponatinib treatment.

Time and dose-dependent effects on K562 cell health following ponatinib treatment.

Time and dose-dependent effects on K562 cell health following ponatinib treatment.

Figure 2A-C. Time and dose-dependent effects on K562 cell health following ponatinib treatment.

That detection of caspase-3/7 activity was correlated with K562 cytotoxicity and was transient in nature, as shown by time course treatment with the other test compounds.

During the evaluation of the highest concentration tested, 100 μM, the kinase inhibitors induced a cytotoxicity increment over time as a result of treatment with the various drugs (Figure 3). As demonstrated by treatment with bosutinib, apoptosis (caspase-3/7 activity) was generally the least detectable in cases of early cytotoxicity (Figure 3A).

Other drug treatments (imatinib, dasatinib, staurosporine) showed increasing apoptosis detection with time that correlated with increased cytotoxicity, as illustrated in Figure 3B-D. The transient nature of caspase-3/7 detection was shown in the case of bosutinib (A) and staurosporine (D) in which the caspase-3/7 detection level peaked early and then decreased with time.

Fold changes in cytotoxicity and apoptosis as compared to an untreated control reveal varied cell health profiles in response to kinase inhibitor treatment.

Fold changes in cytotoxicity and apoptosis as compared to an untreated control reveal varied cell health profiles in response to kinase inhibitor treatment.

Fold changes in cytotoxicity and apoptosis as compared to an untreated control reveal varied cell health profiles in response to kinase inhibitor treatment.

Fold changes in cytotoxicity and apoptosis as compared to an untreated control reveal varied cell health profiles in response to kinase inhibitor treatment.

Figure 3. Fold changes in cytotoxicity and apoptosis as compared to an untreated control reveal varied cell health profiles in response to kinase inhibitor treatment.

When the cytotoxicity data from the apoptosis-inducing compounds was closely evaluated, it showed that when relative fluorescence cytotoxicity assay readings with any dose of compound increased to about 1.4 times the untreated cell control, caspase-3/7 activity was detected within those wells.

Another experiment was performed to determine whether or not a change in cytotoxicity, or cytotoxic threshold, could be set within the Spark® software and employed as a condition for automated injection of caspase-3/7 reagent.

Experiment 2: Using cytotoxicity readout to initiate automated injection of caspase-3/7 reagent

The follow-up experiment was performed to assess Spark’s ® ability to automatically inject caspase-3/7 reagent using a fluorescence cytotoxicity threshold set in the reader software.

A fluorescence reading of the untreated cell control was collected during compound treatment, and a numerical value about 1.4 times the RFU of the untreated control was employed as the cytotoxic threshold for reagent injection for the 72-hour experiment.

As in the earlier experiment, the assay plate was assembled in a similar manner. However, in order to prevent instant automated injection of reagent due to an overt cytotoxic event, the starting concentration of test compound within the dose range was reduced to 20 μM.

After achieving the fluorescence threshold value at the 20 μM concentration, all wells were injected with caspase-3/7 reagent was injected in the dose series. The plate was then incubated for 30 minutes, and luminescence was determined.

Results from Experiment 2

With bosutinib being used as an example, caspase-3/7 activity could be detected at 39 hours across the complete dose series of compound to indicate potent induction of apoptosis when compared to the control (Figure 4).

The viability assay demonstrated that the highest concentration of bosutinib displayed anti-proliferative effects within 10 hours of treatment. Therefore, a decreased caspase-3/7 activity at 39 hours may be due to the decreased number of K562 cells in the well during reagent injection.

This anti-proliferative effect is due to normalizing apoptosis to cell number (viability).

Cytotoxicity-dependent injection of caspase reagent and corresponding viability, cytotoxicity and apoptosis assessment.

Cytotoxicity-dependent injection of caspase reagent and corresponding viability, cytotoxicity and apoptosis assessment.

Cytotoxicity-dependent injection of caspase reagent and corresponding viability, cytotoxicity and apoptosis assessment.

Figure 4. Cytotoxicity-dependent injection of caspase reagent and corresponding viability, cytotoxicity and apoptosis assessment.

The mechanism of action for all tested compounds is summarized in Table 3. Staurosporine and bcr/abl-targeted inhibitors, except for the ponatinib, demonstrated detectable apoptosis during reagent injection.

Ponatinib results correlated with those acquired in Figure 2; cytotoxicity was detected at 14 hours incubation only and an injection of capase-3/7 reagent was triggered in the absence of apoptosis.

Apoptosis may be measured with increased incubation. ionomycin, which is known to cause necrosis and cytotoxicity, did not show apoptosis at five hours.

Table 3. Confirmation of mechanism of action for all tested compounds

Compound [20 μM]

Mechanism of

Action

Time of

Injection

Apoptosis

(Fold Change over

untreated control)

Viability

(Fold Change over

untreated control)

Ionomycin

Calcium ionophore

5 hours

1.1

0.61

Ponatinib

Bcr/abl kinase inhibitor

14 hours

1.2

0.14

Staurosporine

Pan-kinase inhibitor

33 hours

6.8

0.17

Imatinib

Bcr/abl kinase inhibitor

35 hours

12.0

0.32

Bosutinib

Bcr/abl kinase inhibitor

39 hours

5.8

0.16

Dasatinib

Bcr/abl kinase inhibitor

53 hours

7.2

0.36

Conclusion

The data obtained in this article show that the Spark® multimode reader combined with Promega Cell Health Assays supports the determination of mechanistic toxicity with data acquisition for many days. Cytotoxicity and viability could be observed in real-time up to 72 hours during which the lid lifter allowed access to the assay plate for data acquisition and automatic or timed reagent injection.

The interpretation of mode of action can be considerably affected by the window during which apoptosis is determined. If the onset of cytotoxicity is used as a trigger for apoptosis detection, researchers are more likely to locate the window of apoptosis induction specific to their drug and/or dosage, and thus correctly assign the mode of action of the drug.

The initial investigations indicated that changes in membrane integrity correlated with the level of apoptosis detected, and also early detection of cytotoxicity correlated with overtly cytotoxic compounds (1° necrosis) or fast-acting apoptosis inducers.

Further, late detection of cytotoxicity correlated with more potent induction of apoptosis (2° necrosis). The cytotoxicity measurement may be employed to control the time of caspase-3/7 reagent injection. It was found that caspase-3/7 activity at cytotoxic threshold values correlated with apoptosis-inducing compounds.

Acknowledgements

Produced from materials originally authored by Dr Katrin Flatscher, application scientist at Tecan Austria.

Tecan acknowledges Tracy Worzella, Sarah Duellmann, Alisha Truman and Brad Hook of Promega Corporation for performing and evaluating the experiments summarized in this article and presenting the results as a poster on the SLAS.

References

  • RealTime-Glo MT Cell Viability Assay Kit Protocol (TM431, 9/14)
  • CellTox Green Cytotoxicity Assay Kit Protocol (TM375, 5/15)
  • Caspase-Glo® 3/7 Assay Kit Protocol (TB323, 3/15)

About Tecan

Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Apr 4, 2019 at 4:29 AM

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