Written By: Heather Brown-Harding, Ph.D.
When you’re performing imaging, we always want to make sure that any phenotype we see isn’t just an artifact of unhealthy cells or if you’re doing drug discovery, you want to ensure that the treatment isn’t highly toxic to non-target cells. Therefore, it’s important to understand the health of your cells.
To begin to understand if your cells are healthy you need to ask yourself:
- Is the cell still dividing?
- Is the metabolism of the cell changed?
- Are these features of apoptosis or necrosis?
- Are the cells dead or on the verge of dying?
Western Blot and flow cytometry are just 2 of the possible methods for answering these questions, but when spatial or temporal information is important, microscopy is best for cell health.
Since cell health is such a crucial aspect to measure, here are 6 types of assays to help you determine the health of your cells using a microscope.
1. Look for morphology loss – or loss of asymmetry.
Use bright field imaging to determine whether your cells are spread out – do they look as they normally would in cell culture? This is the simplest way to see if your cells are “unhappy.”
If they do not look as they do in culture, what is different?
Have they rounded up? Is there blebbing?
Imagine the cells like the jelly blobs in a lava lamp.
Loss of membrane symmetry is the first sign that apoptosis is occurring, but blebbing is a sure sign that your cells will die shortly. To prevent this, check–and lower if necessary–the concentrations of drugs, and make sure that your carrier (i.e. your solvent – DMSO, ethanol, etc.) is less than 1:1000.
2. Measure proliferation.
This is as simple as performing a growth assay to compare the number of cells over time.
You can utilize proliferation markers such as Ki67 staining or incorporation of the nucleoside analog, such as BrdU. This will tell us if the DNA is still replicating.
Why is this important? All signs may point to your cells being alive, but they might not be healthy enough to replicate. Cells under stress do not proliferate because it takes a lot of energy to do so. If you are developing a drug that is tolerated but prevents cellular proliferation, then you are likely to have severe side effects. For example, you don’t want to develop an antiviral that inhibits white blood cell proliferation or tissue repair.
3. Measure caspase cleavage.
Caspases are one of the main mediators for apoptosis. Caspases are translated as pro-caspases and are considered “inactive zymogens.” Caspase 3 undergoes a cleavage event becoming a protease which mediates DNA condensation, DNA fragmentation, and cell blebbing.
Caspase cleavage can be monitored with western blot, microplate reader, flow cytometry, or microscopy. Pick whatever method you have access to, but for microscopy, staining for active caspase 3 and measuring intensity will give a good readout of the beginning of apoptosis.
Caspase activation is easier to visualize for beginners than membrane asymmetry, so it a great assay to determine if your cells are at just starting to become apoptotic. Some intracellular microbes will activate caspase, but if the infection is cleared the cells can still survive. For this reason, caspase cleavage is usually paired with a TUNEL assay (see below).
4. Determine the localization of Cytochrome C.
Cytochrome C is a protein that is localized to the inner mitochondrial membrane and is critical in the electron transport chain. Cytochrome C is released with the integrity of the mitochondria is compromised, so it is a good measure of cell health
This assay is very similar to measuring caspase and you can do it via Western Blot (with mitochondrial enrichment) or immunofluorescence to determine the localization of cytochrome C. Immunofluorescent assays are generally produce cleaner data since you don’t have to do the extra mitochondria enrichment/isolation.
Fluorescent Cytochrome C assays are as easy a measuring localization to the mitochondria.
If you’re interested in performing cytochrome C release in real-time, a nanosensor has been developed for live-cell imaging.
5. Measure mitochondrial membrane potential.
TMRE is a red-orange dye that accumulates in the mitochondria of healthy cells. A decrease in fluorescence would, with the use of TMRE, indicate that a cell that has lost mitochondrial membrane potential. Without membrane potential, ATP cannot be produced, and mitochondria can no longer act as cellular powerhouses.
Another useful dye is JC-1, a cationic green-red dye that exhibits a potential-dependent accumulation in the mitochondria. Mitochondrial depolarization is indicated by a decrease in the red-to-green fluorescent intensity ratio, so this is great for quantitative imaging. This ratiometric measuring compensates for any difference in cell uptake between experiments. If you’re dealing with non-adherent cells, JC-1 is also ideal for flow cytometry.
Finally, it’s important to include a control to ensure you can accurately measure any changes in membrane potential. A commonly used positive control is FCCP which is an ionophore uncoupler of oxidative phosphorylation. Treating cells with FCCP eliminates mitochondrial membrane potential which makes FCCP a very good positive control for these types of studies.
6. Perform a TUNEL assay.
Most people are familiar with the TUNEL assay, as it predicts the coming end of a cell. The TUNEL assay visualizes DNA fragmentation, and it can be performed as either a colorimetric assay, such as DAB, or a common fluorescent assay. Both DAB and fluorescent assays can be analyzed with a microscope.
Commonly, I find users will use the colorimetric assay on tissues and the fluorescent assays on cells. Why is this? Because tissues have much higher background auto-fluorescence, so using a colorimetric assay will give you fewer problems when you’re trying to see the signal through the background – you are not exciting the sample with fluorescence.
When performing microscopy experiments, you need to be aware of the health of your cells. This is how you can be confident in your results and know that you are not just seeing artifacts of unhealthy cells. Artifacts are a big problem in microscopy, and you don’t want to waste your time following an incorrect lead or having a reviewer question whether the phenomenon is real. A few microscopy assays that you can use to understand cell health are looking for morphology loss or loss of asymmetry; measure proliferation; measure caspase; determining the localization of cytochrome C; measuring mitochondrial potential; and performing the TUNEL assay.
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In her free time, she enjoys playing with her cat & dog, trying out new craft ciders and painting.You can find her on twitter (@microscopyEd) a few times of day discussing new imaging techniques with peers.
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