Multi-Color Caspase 3/7 Assays with Mica

From imaging to analysis

Two-color caspase assay with tile scan Two-color_caspase_assay_with_tile_scan.jpg

Caspases are involved in apoptosis and can be utilized to determine if cells are undergoing this programmed cell death pathway in so-called caspase assays. These assays can be run by e.g. flow cytometry, plate readers, or on a microscope which adds visible, structural information to the quantified data. In this article we describe how Mica can be used to perform caspase 3/7 assays. With the help of tools like the Navigator or Pixel Classifier, Mica makes it easier to set up, execute and analyze caspase 3/7 assays, even for inexperienced users.

Image: Two-color caspase assay with tile scan. U2OS cells were treated with the nuclear marker DRAQ5 (magenta) and CellEvent™ (yellow). 4 mM staurosporine was added to induce apoptosis. A tile scan of 2x2 FOVs was acquired every 30 minutes for 16 hours duration with the two fluorescence channels using a 20x objective.

Introduction

Apoptosis or live/dead assays are used to specify the toxicity or effectiveness of certain substances by applying them to living cells and watching the reaction. Increasing death rates over time or dose-dependent death rates indicate an effect. One prominent example is to determine the anti-cancer effect of potential drugs. 
The apoptotic status of cells can be determined with commercially available dye kits. These kits are based on fluorescent dyes which are able to mark live or dead cells. 
One category of apoptosis assays are caspase assays. Caspases are cysteine-aspartic acid specific proteases, involved in apoptosis. They are also utilized to differentiate caspase-mediated apoptosis from necrosis.
The dye kit, which was used here, utilizes a DNA-binding reagent whose fluorescent behavior is blocked by a four amino-acid peptide (DEVD). Once caspase-3 and -7 (caspase-3/7) are activated, which is the case when cells undergo apoptosis, they cleave the DEVD peptide and the DNA-binding reagent starts fluorescing (see video 1). 

Video 1: Principle of the caspase 3/7 assay: COS7 cells were treated with the nuclear marker DRAQ5 (magenta) and with CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen™) (yellow). CellEvent™ is a DNA binding dye which starts fluorescing after a four-amino acid peptide (DEVD) is cleaved by caspase 3/7. Thus, it can detect cells undergoing caspase 3/7 mediated apoptosis. Staurosporine was added to induce apoptosis. Images were acquired every 20 minutes for 12 hours duration both in brightfield and with the two fluorescence channels using a 63x objective. After a while, cells undergoing apoptosis can be identified by yellow fluorescence.

Challenges

Experiments with many varying parameters, e.g. concentrations of additives, different incubation times, types of staining, cell lines, etc., are commonly done in multi-well plates. Besides the advantage of having many different experimental conditions in one reaction vessel, the other benefit is that very small volumes of reagents and low numbers of cells can be used.
However, the number of different wells and experiments can also confuse users during execution.
The Navigator tool on Mica can be used to keep the overview when setting up a well-plate experiment. This includes, for example, tile-scans or time-lapse experiments in each single well which can be planned and set up on a virtual canvas (see figure 1).

Tracking cellular events requires spatiotemporal correlation of the single fluorescence channels which were imaged. Conventional widefield microscopy commonly records one channel at a time. So, every cellular structure is recorded sequentially one after another. This means that two different structures are recorded at two different time points. For very fast cellular events, this can make a difference, especially for colocalization studies. 
Simultaneous imaging circumvents this disadvantage by recording all fluorophores at the same time. In the case of a caspase assay, this means that users can watch, for example, how mitochondria react at the moment caspase-3/7 are activated.
Mica can even detect 4 fluorophores simultaneously, enabling users to observe another cellular structure, concurrently, in addition to nuclei, caspase-3/7 activity, mitochondria, etc. (see figure 5).

In the end, a caspase assay needs to be quantified if the aim is to determine how efficient a certain reagent is at inducing caspase-mediated apoptosis. This quantification can be achieved e.g. with a dedicated external software. 
Instead of using a second software, Mica has an onboard analysis solution. With the help of the integrated Pixel Classifier, users can mark some regions of interest (ROI) which are used by the AI algorithm to detect all other ROIs (see figure 2). In the case of a caspase assay, the nuclei signal can be used to determine the overall quantity of cells. The CellEvent™ signal can be used to identify caspase-mediated apoptotic cells.

Methods

For the assays of this case study, U2OS or COS7 cells were seeded on 96-well plates and grown over night. On the day of the experiment, living cells were incubated with DRAQ5, respectively SPY-650-DNA, and TMRE for 15 minutes. Afterwards, the medium was exchanged, and cells were incubated with CellEvent™ for the rest of the experiment. The apoptosis inducer staurosporine was added to each well (3 µM – 7 µM).

For the four-color caspase assay, U2OS cells were seeded on 96-well plates and grown over night. On the day of the experiment, living cells were incubated with DAPI and TMRE for 45 minutes. Afterwards, the medium was exchanged, and cells were incubated with CellEvent™ and SiR-Tubulin for the rest of the experiment. The apoptosis inducer staurosporine was added to each well (3 µM – 7 µM).

Live-cell imaging was done with Mica for the indicated time intervals and duration at 37°C, 5% CO2, and ~65% humidity. Assays were set up and executed with the Navigator tool. For some experiments, tile scans were run (refer to video 2). For tile scans, the focus strategy “Focus Map” was used, otherwise “Keep Focus” was utilized to keep cells in focus over the time-lapse experiment.

Video 2: Tile-scan movie of a three-color caspase assay. U2OS cells were treated with the nuclear marker SPY-650-DNA (blue), mitochondrial membrane potential marker TMRE (magenta) and with CellEvent™ (yellow). 5 mM staurosporine was added to induce apoptosis. A tile scan of 2x2 FOVs (fields of view) was acquired every 15 minutes for 12 hours duration both in brightfield and with the two fluorescence channels using a 20x objective. While Caspase activity increases over time, mitochondrial activity decreases.

For the experimental data analysis, the Mica onboard analysis function was used. One of its central components is the AI-based Pixel Classifier which can be found in the “Learn” tab. 
With the Pixel Classifier, users can mark a region of interest (ROI) which serves as a model for all other regions to detect. For the case of the example shown in figure 5, nuclei were marked and detected. The same was done for the mitochondria-activity marker TMRE and caspase-positive cells. Afterwards, the batch of images was analyzed across the whole time-lapse. 
After computation, the results can be displayed in the “Results” tab as a Scatter Plot, Colocalization, Histogram, Pie Chart, Box Plot, or Time Series.

Results

Quantification of the two cell dyes SPY-650-DNA (nuclei) and CellEvent™ (caspase 3/7 activity) sheds light on cells which undergo caspase-mediated apoptosis during the live-cell experiment. The number of nuclei (here the parameter “Area” was chosen as an analogy) gives information about the number (resp. area) of cells per image and can be correlated with the number (resp. area) of cells undergoing apoptosis accordingly. Imaging of the additional marker TMRE (mitochondria membrane potential) sheds light on the activity of mitochondria. 

Quantified data (e.g. Length, Width, Area) is shown in a table below the acquired images and can be exported in Excel format. The acquired images can be viewed for every single time point of the time-lapse imaging and can be overlaid with the identified ROIs.
The graph of the three-color caspase 3/7 assay (Figure 4) shows that the nuclei signal rises in the beginning and declines later. The rise can be explained by the fact that the nuclear stain must bind to the DNA which takes some time. On the other side, the decrease of the SPY-650-DNA signal is due to disintegration of the nuclei which can be observed in the associated images.
The other signals either increase (CellEvent™) or decrease (TMRE) over time: whereas there are more caspase-mediated apoptotic cells, the number of active mitochondria goes down.

Mica enables users to image 4 fluorophores at one time. For the case of a caspase 3/7 assay, this helps investigating the fate of an additional cellular component during apoptosis – with absolute spatiotemporal correlation. For the example shown in figure 5, the actin cytoskeleton (SiR-Actin) was stained in addition to the nuclei (DAPI), active mitochondria (TMRE), and caspase-active cells (CellEvent™). 
At higher magnification (63x), users can watch the actin cytoskeleton collapsing upon caspase activation. At the same time, the nucleus granulates and mitochondria stop operation. Since all channels are acquired with one exposure, the imaged cellular events can be precisely correlated. 

Conclusions

Caspase assays give insights into the anti-cancer potential of drugs. For this purpose, living cells must be differentiated from apoptotic cells with spatiotemporal precision. 
For statistically reliable results, increased throughput is necessary. That is why such experiments are executed in well-plates and must be quantified.
Mica meets all the requirements above. With FluoSync, up to 4 different fluorescent dyes can be imaged simultaneously, from a single petri dish to 96-well plates. With the integrated incubation system of Mica, living cells can be cultured for days and the AI based on-board analysis function helps users to acquire reliable data.

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