Using non-invasive live-cell imaging to improve standard biological assays
Cellular assays are used to assess cellular proliferation, metabolic activity and viability, immunological responses, DNA damage and expression of delivered constructs. However, the assays that are generally used to measure these parameters are end-point measurements. Any kinetic information is either lost or, to investigate temporal effects, many cell cultures have to be set-up in parallel and sacrificed at all time-points of interest. As such an alternative method could be useful to support or replace these end-point cellular assays.
Adapting non-invasive live-cell imaging of culture plates in a multi-well format can allow researchers to compare the temporal effects of multiple treatments and/or conditions simultaneously. In the article below, we will discuss how using non-invasive live-cell imaging compares to traditional cellular assays.
Advantages of non-invasive live-cell imaging
Time-lapse imaging allows researchers to assess cellular characteristics at multiple time-points, enabling kinetic analysis. For instance, researchers can assess time-dependent changes in population growth rates, cellular density and colony formation. The ability to pinpoint important events during experimental incubation periods like attachment and detachment, cell death and cellular proliferation, overcomes issues with making assumptions about the state of the cell culture.
Improving cellular proliferation assays using live-cell imaging
The bromodeoxyuridine (BrdU) assay is typically used to measure cellular proliferation. This is determined by measuring BrdU incorporation into DNA using a monoclonal antibody specific for BrdU which can be detected using immunolabeling techniques like immunofluorescence or enzyme-linked immunosorbent analysis (ELISA) assays, but the reagents utilized in this technique are expensive, and optimization is required, since the rate of BrdU incorporation depends on the proliferation rate of individual cell lines. Furthermore, this method requires harsh conditions, such as permeabilization, fixation, and DNA denaturation, to facilitate antibody binding to genomic BrdU 1,2.
Another method used to analyze proliferation, is to measure the amount of nuclear proteins, including Ki67 (not detected in resting cells) and proliferating cell nuclear antigen (PCNA), which are present in high concentrations during the M, G2, and S phases of mitosis. Here immunofluorescence, immunohistochemistry, or western blotting are used, but these data-sets are difficult to quantify and only reveal cells that are proliferating at the time of collection 1.
Other techniques to assess cellular proliferation involve the use of dyes. For instance, carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) is a non-fluorescent, cell permeable dye that can be cleaved by intracellular esterases into CFSE to emit green fluorescence 3. This compound is used for labeling the cells, so, with each cell division, fluorescence decreases 4. Traditionally, CFSE incorporation has been measured using fluorescence-activated cellular sorting (FACS), but fluorescence can only be monitored for up to 8 divisions before the signal decreases to background levels 4. In addition to CFSE staining, cellular fluorescent staining using flow cytometry can be performed using other dyes, such as propidium iodide (PI), which is used to detect dead cells and intercalates into DNA 2. However, PI staining requires nuclease treatment, since the dye cannot differentiate between DNA and RNA 5.
The proliferation assays described in the above are all endpoint assays and only assess the cells proliferating at the time of collection. Since live-cell imaging does not require fixation or permeabilization7, utilizing live-cell imaging in combination with the aforementioned proliferation assays can enable researchers to pinpoint the exact timing required for cells to reach the target confluency for experimentation, reducing time required for optimization.
Live-cell imaging can analyze the average proliferation rate of the same cellular population when images are obtained at multiple time-points, allowing analysis of kinetic vs. end-point measurements 8. Non-invasive live-cell imaging does not have to be an alternative for performing cellular proliferation assays, rather it can be used to support these traditional assays. In addition, non-invasive live-cell imaging allows researchers to analyze the proliferation of a variety of cell culture models, including three-dimensional models, such as organoids, which is difficult to assess, since these cultures are delicate and are typically grown in suspension.
One particular assay where live cell imaging can be useful is the colony formation assay. This assay determines a cell’s ability to form a colony over a long period of time in culture 6. However, colony counting and assessment of colony sizes are primarily done manually, which can be a tedious process 6. To support researchers specific software developed for live-cell imaging enables quantification of colony formation (colony size and colony count) over time, reducing manual labor. Time-lapse video microscopy captures sample heterogeneity that might otherwise be obscured in manual colony analysis. A multi-well format facilitates assessment of multiple treatments and/or conditions simultaneously.
Analyzing cellular viability using metabolic activity or live-cell imaging
To screen the effects of test compounds on metabolic activity as an indicator of cellular viability, the MTT assay is typically performed. In this assay, the tetrazolium dye MTT 3-(4,5-dimethlthiazol02-yl)-2,5-diphenyltetrazolium bromide is reduced by cellular NAD(P)H-dependent oxidoreductases to an insoluble formazan, which has a purple color 9. The absorbance of this dye is then measured using a spectrophotometer at 570nm. However, this assay is unsuitable for cells grown in suspension, has to be optimized for cell density, and may not be utilized with compounds that interfere with absorbance at 570 nm 9. Furthermore, this assay may give false positive results when metabolism is affected, since it cannot differentiate between cell death and cell cycle inhibition 10.
Another enzymatic assay typically used to assess cellular viability is the LDH assay. In this assay, L-lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to L-lactate and NADH to NAD+ during glycolysis and the reverse reactions during the Cori cycle 11. Cellular damage and/or exposure to insults stimulates LDH release into extracellular medium 12. Released LDH can be detected using a colorimetric assay, wherein iodonitroterazolium (INT) is converted into a red color formazan; absorbance of formazan can be quantitatively measured using a spectrophotometer at 490nm 12. However, serum, routinely used as a cell culture reagent, and other compounds have inherent LDH activity, thus requiring experiments to be done in serum-free or low serum conditions, which can induce epigenetic modifications 13.
Using live-cell imaging to monitor confluency can reduce experimental optimization time for these assays, since cellular density is critical for both the MTT and LDH assays. To measure the health of the cells before or during a metabolic assay, live-cell imaging can be used to assess morphological changes due to necrosis or apoptosis 6. Time-lapse imaging ensures the MTT and/or LDH assays are performed at an optimal time period for all of the experimental conditions being tested. Furthermore, live-cell imaging may be adapted to analyze the effects of compounds that are unsuitable for MTT and LDH colorimetric assays on cellular viability, due to interference with absorbance spectra. .
Using live-cell imaging to assess immunological responses
To measure immunological responses, immune cell proliferation, cytotoxicity, and cytokine production are measured 14. For instance, the ability of T cells to proliferate in response to an antigen has been used to indicate the presence of antigen-specific CD4+ helper T cells. In this assay, the purified T cell or peripheral blood mononuclear cell (PBMCs, fraction of lymphocytes that consists of T, B, and NK cells) samples are mixed with antigen or antigen in the presence of HLA-matched antigen presenting cells and then proliferation is assessed using endpoint assays 14.
In immune cell killing assays, immune cells of choice (T cells, NK cells, or PBMCs) are co-cultured with target cells, and cell death can be measured using various endpoints, such as annexin V staining or caspase3/7 staining 15. Furthermore, to test cellular and humoral immune responses, endpoint ELISA, enzyme-linked immunospot (ELISpot), and flow cytometric assays assessing IFNγ production and/or MHC tetramers are typically performed after antigen exposure 16.
However, since experimental studies can be limited by the amount of blood obtained from experimental subjects, and immune cells should be used immediately after isolation 17, using live-cell imaging to analyze to assess immune cell proliferation and cytotoxicity during the course of an experiment prevents researchers from missing critical time points due to flaws in experimental design.
For researchers looking to receive email alerts when the cells have reached the target confluency, we recommend the CytoSMART Lux2.
Assessing DNA damage using live-cell imaging
To assess DNA damage, the terminal dUTP nick end-labeling (TUNEL) assay has been widely used. In this assay, terminal deoxynucleotidyl transferase (TdT) adds random nucleotides to DNA fragments produced during apoptosis, and the resulting dUTP can be labeled with probes for detection using fluorescence microscopy or a colorimetric assay 18. This assay is routinely used in immunohistochemistry for studying DNA damage in tissues. However, it is expensive, may not be suitable for large-scale analyses, and cannot quantify the magnitude of DNA damage in a single cell, rather the number of cells in a population with DNA damage is quantified 18.
Another method routinely used to assess DNA damage is the comet assay or single-cell gel electrophoresis. In this assay, a break in DNA results in relaxation of supercoiling, and migration of cleaved DNA out of nuclei in an electric field results in the formation of a “comet” tail, so analysis of the DNA “comet” tail and nucleoid shape is used to assess DNA damage 19. However, this assay is difficult to calibrate, cannot detect mitochondrial DNA damage, and is difficult to standardize 20. Both of these assays are also endpoint assays, only allowing measurement of DNA damage at the time of collection. In contrast, live-cell imaging can enable researchers to visualize and quantify DNA damage and repair over time in single cells 21.
Non-invasive live-cell imaging can improve standard biological end-point assays assessing - cellular proliferation, metabolic activity and viability, immunological responses, and DNA damage - by preventing researchers from missing critical time points during experiments when cultured cells are proliferating and/or dying.
The ability to observe live cells in a multi-well format allows researchers to compare the effects of different treatments and/or conditions simultaneously. Moreover, composite recordings of what is occurring in a single well negates the need for researchers to manually compile data from randomly selected areas of interest to assess the effects of a particular treatment and/or condition. Additionally, since some live-cell imaging microscopes can be placed directly in CO2 incubators or hypoxia chambers, researchers can also assess the effects of experimental treatments and/or conditions without setting foot in the lab and even receive email alerts when cells have reached the target confluence for passaging or experiments
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