Live Cell Studies

Introduction

Analyzing live cells can be an advantage or a necessity, depending on the application. On iGeneration platforms, live-cell analysis is easily accomplished with carriers such as petri dishes and multi-well plates. A wide variety of live-cell assays are compatible with iGeneration systems. This discussion highlights a selection of these applications.

Highlights of Analysis with iGeneration Imaging Cytometers

There are many fluorescent stains available that allow measurements of cell cycle, metabolism, apoptosis, and necrosis in unprocessed, or “live“ samples. The diversity of probes includes markers for reactive oxygen species, mitochondrial membrane potential, and nucleic acids.

With Laser Scanning Cytometry, it is possible to measure the amount of these markers and compare the differences within a heterogeneous population or between treated populations.

Apoptosis Assay

Drug titration studies for apoptosis detection can be performed using multi-color analysis. Control and drug-treated wells are treated with Hoechst, Yo-Pro™ (Molecular Probes) or AnnexinV-FITC, propidium iodide (PI) and, if desired, a mitochondrial membrane potential marker such as MitoShift™ (Trevigen).

In this assay:

Normal cells are detected by Hoechst and MitoShift™ staining.
Apoptotic cells are detected by Yo-Pro™ or AnnexinV-FITC staining.
Necrotic cells are detected by PI staining .

This laser-scanned field shows a mixed population of cells including normal (blue only), apoptotic (blue and green) and necrotic (red or white) cells.

Using a scattergram that resolves cells based on their green vs. red fluorescence, apoptotic cells (green dots) can be resolved from necrotic cells (red dots) and healthy cells (black dots). The accompanying histogram plots the cells based on the apoptotic marker.

Biomarker FRET Assay

iGeneration instruments can detect Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP). When these proteins are in close proximity, such as when they are linked by a small peptide, excitation of CFP only (405 nm light) can result in fluorescence resonance energy transfer (FRET) when the emitting light energy is captured by the YFP (which results in fluorescence energy emission from YFP).

Since YFP is not excited by short-wavelength light, any fluorescence detected that is YFP-specific (i.e., its emission wavelength) results from FRET. A FRET biosensor was created linking CFP and YFP with a peptide sequence based on the cleavage site in caspase-3. When this construct is expressed in healthy cells, FRET can be measured. During apoptosis, this biosensor will be cleaved and measurable FRET is decreased when caspase-3 is activated.

Data provided by Dr. Natasha Barteneva, M.D., Ph.D., The CBR Institute for Biomedical Research, Boston, Massachusetts, USA.

The following images compare absence of biomarker FRET (left) and detectable biomarker FRET (right).

The following windows show cell populations exhibiting FRET before drug-induced apoptosis (Left) and measurably decreased FRET following drug-induced apoptosis (Right).

In an analysis where drug titrations leading to varying levels of caspase-3 activation are used, the FRET biosensor measurements can be plotted using a statistical analysis that shows the dose-dependent response.

Untreated control wells are shown in the three histograms on the bottom right.

Viability

With appropriate markers, cell viability can be measured on iGeneration systems. In the laser-scanned image example below, viable cells are detected by the presence of calcein green. Only healthy cells will retain this marker. The cell positions can be correlated with the accompanying laser scatter image. In this case, all cells have acquired the viability marker.

Fluorescent Proteins

Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) are readily detected with iGeneration systems. CFP requires the violet laser, while GFP and YFP are detected using the Argon laser for excitation.

Membrane Potential

A variety of membrane-potential fluorescent markers are commercially available. Markers excited at 405, 488 or 633 nm are compatible with iCyte and iCys. A particularly interesting example is MitoShift ®. This compound accumulates in mitochondria that have a normal membrane potential. When the membrane potential is lost during apoptosis, the change can be monitored with iCyte or iCys.

Phagocytosis, Endocytosis, Internalization Assays

iCyte or iCys can be used to detect and measure the accumulation of fluorescence in cells. Using appropriate fluorescent markers on compounds, particles, or organisms, the accumulation can be monitored in a number of different ways, from counting the internalized particles to measuring the internalized fluorescence. Examples include the phagocytosis of fluorescently labeled bacteria to the pinocytosis of small fluorescent compounds.

Kinetic Assays

iGeneration systems can monitor the same area in a well over time, allowing study of kinetic changes in the studied population. Examples include changes in membrane potential markers and fluorescence uptake (e.g., phagocytosis or endocytosis).

Cell Spreading

The area and circularity of cells may be measured on the iCyte or iCys, allowing for detection of the morphological changes that occur as cells go from small round structures to larger irregular morphologies.

Benefits of Using iCyte or iCys Technology

Multiple lasers and detectors allow complex assays requiring multiple markers. Using advanced data processing, cells may be identified for analysis using either fluorescence characteristics or the cells’ ability to absorb or refract light. Using carriers such as 96-well microtiter plates can facilitate both sample processing and throughput. Multiple probes can be analyzed in a single sample while obtaining widefield images, which may be correlated to the quantitative data in field images. iGeneration systems combine the advantages of flow cytometry with those of image analysis systems. The iCys and iCyte inverted format supports a diversity of carrier types, allowing flexibility in matching the needs of your application with the appropriate carrier type.

More Information

For more information, review the CompuCyte Published Materials and CompuCyte Bibliography.
Request the FRET Assay Application Note.