Leave a Reply Cancel reply Enter your comment here Fill in your details below or click an icon to log in:. Email required Address never made public. Name required. Create your website with WordPress. Follow Following. Travis Thuo. Sign me up. Already have a WordPress. This chapter will describe common methods for measuring dead cells in culture using a plate reader that can be applied to high-throughput screening.
This is an introductory review focused on the most frequently used methods for measuring the number of dead cells using a plate reader. We will describe basic assay methods and factors to consider when choosing an assay as well as the advantages and disadvantages of different methods. A separate chapter will describe HTS assays to measure apoptosis. For all assays using cultured cells as a model system, it is valuable to know how many live and dead cells are present during or after the end of the experiment.
This becomes especially important when cells are incubated for a period of time adequate to enable growth and division and thus change the total number of cells present. Relating live and dead cell numbers by a normalization process improves the statistical robustness of the assay. However, the use of cell number as an internal control is often overlooked in many cell-based assays such as reporter assays to detect expression of stress response genes or testing for other stress related events.
A frequent use of cells in culture is for a commonly used cytotoxicity assay where cells are exposed to a test compound and after some period of incubation, a marker is measured to reflect the number of viable cells present compared to positive toxin and negative vehicle control treatments.
In addition to estimating the number of live cells, it can be of great value to measure the number of dead cells that have accumulated over the course of the experiment and to be able to distinguish between cytotoxicity and cytostasis or growth arrest. In some cases, estimating the number of accumulated dead cells may be more sensitive than measuring a decrease in a marker used to estimate viable cell number.
The two commonly used methods of estimating dead cells take advantage of the loss of membrane integrity and the ability of indicator molecules to partition into a compartment not achievable if the cell membrane is intact.
As illustrated in the diagram in Figure 1 , assays used to detect dead cells include measuring the leakage of a component usually an enzyme marker from the cytoplasm into the culture medium or the penetration of an otherwise non-permeable dye into cells with a compromised membrane. This chapter will describe options for both general approaches for measuring dead cells.
Illustrates scanning electron micrographs of isolated rat hepatocytes. The left image is meant to depict an intact live cell and the image on the right depicts a dead cell with a damaged membrane. The loss of membrane integrity enables leakage of dead more The selective staining of dead cells with trypan blue and microscopic examination on a hemocytometer is one of the most frequently used routine methods to determine the cell number and percent viability in a population of cells.
The general concept is that trypan blue is excluded from live cells, but penetrates dead cells with a damaged plasma membrane. Longer incubations with solutions of trypan blue may result in faint staining of the viable cells in the population, possibly due to slow uptake of dye molecules.
The mechanism of selective staining of dead cells may actually involve impermeability of aggregates of trypan blue 1. There are several sources for published protocols or instructional videos describing the use of trypan blue and the many details associated with correctly using a hemocytometer 2. Counting cells using a hemocytometer. Counting of cells using Trypan Blue and a haemocytometer.
The trypan blue staining technique is usually performed on a single sample such as when passaging a stock culture flask of cells or relatively small numbers of samples from simple experiments. The main disadvantages of this technique are: the error involved with measuring a single sample, the subjective judgement of the user to determine what is a dead cell or stained debris, inconsistency among operators, and the time and manual labor involved with measuring multiple samples 3. There is a large number of nucleic acid binding dyes that can be used to stain cells for microscopy or flow cytometry but have limitations for use with assays using plate readers to detect signal.
Many dyes have somewhat similar properties which make it difficult to choose the most appropriate probe for a particular purpose. A general description of many of the nucleic acid binding dyes can be found in the following link to The Molecular Probes Handbook found on the ThermoFisher Scientific website. There are many fluorescent DNA binding dyes to select from which are generally considered to be non-permeable to viable cells and can be used for detection of the accumulation of dead cells in culture using a multi-well plate format; however, there are a variety of factors to consider when selecting the most appropriate dye for assay development.
The most important and practical factors to consider when choosing a dye include: the emission wavelength, selectivity for staining DNA, cell permeability, solubility at the vendor-recommended concentration, detection sensitivity and cytotoxicity.
Fluorogenic DNA dyes that readily pass through the intact cell membrane and stain the nucleus of live cells should be avoided for consideration for measuring dead cells. Chiaraviglio and Kirby 4 have recently reported on the evaluation of non-permeable DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high-throughput screening format. They include a useful table summarizing the properties for many nucleic acid binding dyes as well as demonstrating the ability to multiplex fluorescent dead cell assays with other cell-based methods.
An important factor to consider when choosing any fluorescent dye is the emission wavelength spectrum. Knowledge of the excitation and emission spectra and the extent of any spectral overlap can be used to predict compatibility of two fluorescent assays and to select an appropriate filter set to avoid overlap of emission of different fluorophores. For example, a green-emitting DNA binding dye would be a logical candidate to multiplex with an assay detecting a red fluorescent protein.
A table listing filter sets used for many of the nucleic acid binding dyes is included in the Chiaraviglio and Kirby reference 4. The ability to multiplex more than one fluorescent or luminescent assay provides flexibility during assay design. Measuring the number of dead cells is often used as a multiplexed internal control for other cell-based screening assays. Sequential multiplexing by recording data from a fluorogenic assay prior to addition of a second luminogenic assay chemistry can expand the possibilities for multiplexing.
The DNA binding dyes can be considered to be environmental sensors, meaning they change fluorescent properties after binding to various molecules. The various nucleic acid binding dyes may exhibit between a to fold increase in fluorescence upon binding to double stranded DNA.
That fold-increase can contribute to the relative sensitivity of detection of the number of dead cells. In most experimental conditions using a growing population of cultured cells in vitro , the quantity of DNA is proportional to the total number of cells present; however, changing culture conditions to induce rapid cell growth, to starve cells of nutrients, or induce differentiation to result in multinucleation may have a greater influence to change the amount of nucleic acid present in cells.
The ideal situation for quantitatively detecting dead cells is for the dye to selectively bind to double stranded DNA. If the dye binds to double stranded RNA which may change under stimulatory or stressful culture conditions, using dyes that also bind to RNA can lead to artifacts and misinterpretation of results. Even slight adverse effects of DNA binding dyes can limit their usefulness for real-time assay protocols where the dye is exposed to cells for extended periods of time.
Dyes that cause cytotoxicity upon long term exposure to cells may be the result of partial permeability. Membrane permeability may depend on the cell type, the overall health of the cells or whether the dyes are substrates for efflux pumps that result in expulsion from the cytoplasm even if the dye does enter the cell.
Reagent toxicity is not a problem if the dye is going to be used to stain cells for an endpoint assay protocol; however, cytotoxicity is critical to consider if cells will be cultured in the presence of the dye for an extended period to perform a real-time assay. The use of DNA binding dyes for long term real-time detection of the accumulation of dead cells must consider if there is any influence of the assay reagent on the health or responsiveness of the cell model system.
For example, for some cell viability assays, it is well known that reagents to estimate viable cell number e. MTT and resazurin can be toxic to the population of cells, even during a few hours of exposure 5. Similar reagent cytotoxicity effects are known for the DNA binding dyes. Figure 2 shows the effects of three different DNA binding dyes continuously exposed to four different cell types for 72 hours before measuring cell viability using an ATP assay. Effect of DNA binding dyes on cell viability.
Four different cell types were treated with the vendor recommended concentration of DNA binding dye and cell viability assayed at various times up to three days using ATP content as the marker.
The data suggest similar nucleic acid binding dyes may have different effects on cell viability and those effects can be cell type specific. Understanding the mechanism and use of these DNA dyes is therefore important to determine the best probe for the desired assay design.
Like all potential toxins, the cytotoxic effect of assay reagent components can be expected to depend on the concentration, the duration of exposure, and the susceptibility of individual cell types.
Appropriate controls vehicle only without dye are recommended to validate each dye and cell type combination to determine if there is a cytotoxic effect of the assay reagent. It is suggested to use the vendor recommended concentration as a starting point and test a range of concentrations of dyes with each cell model system to confirm there is not an artefactual cytotoxic or cytostatic effect. Several examples of DNA binding dyes classified as nonpermeable to live cells are commercially available.
The following example protocols are based on using the CellTox Green Cytotoxicity Assay which contains a non-permeable asymmetric cyanine dye which binds the minor groove to stain DNA of dead cells.
The CellTox Green Dye is optimally excited at nm with a peak emission at nm. For a complete detailed description of protocols including: reagent preparation, determination of linear range, detection sensitivity for individual cell types, and endpoint or homogeneous assay protocols, refer to Promega Technical Manual The general goal of the protocol is to create a dilution of the DNA binding dye in the sample of cultured cells to be measured.
There are optional protocols to prepare a working dilution in a balanced salt solution Assay Buffer and achieve a final dilution of dye depending on whether you choose to run the assay in a real-time mode record data periodically over days in culture or an endpoint mode measure once at the end of the experiment. That approach achieves a dilution of dye in culture medium and allows for addition of test compounds or vehicle control as a 2X preparation in an equal volume of culture medium.
Mix the tube containing the suspension of cells by inversion or gently vortex to ensure dye homogeneity prior to seeding into assay plates. Seed cell suspension in opaque walled assay plates to avoid fluorescent signal crosstalk with desired volume and cell number.
Add test compound treatment and vehicle controls to each well using a volume of medium equivalent to that used to seed the cells so the final concentration of dye is a dilution of the original component.
The following example in Figure 3 illustrates results from a real-time assay showing an increase in fluorescence with both higher Terfenadine concentration and longer incubation time. The increase in fluorescence indicates the accumulation of dead cells over a three day incubation. HepG2 cells treated with various doses of Terfenadine. CellTox Green Dye was added at time of Terfenadine dosing. Fluorescence was measured every hour over a three day incubation in a plate reader with an environmental chamber to control temperature at more Addition of DNA binding dye can be used as an endpoint assay at the end of an experiment to stain dead cells and estimate how many are present.
To assist in mixing of reagent and to avoid pipetting extremely small volumes, it is convenient to create a dilution of dye in Assay Buffer and add a vol:vol ratio of diluted dye reagent to the sample of cells in culture medium. The DNA binding dye in CellTox Green is functionally inert to the viable cell population which enables subsequent measurement of other parameters using a variety of compatible assay chemistries.
One limitation to consider during multiplex assay design is the total volume available in the sample well. If you are performing a multiplex assay that requires addition of other reagents, you can include the DNA dye in the suspension of cells before dispensing into the assay plate or you can add a smaller volume of a more concentrated DNA dye reagent as long as the final concentration in the assay well is of the original dye component.
For any lytic endpoint assay that will result in the second multiplexed reagent lysing the cells, the fluorescence value corresponding to the number of dead cells must be recorded first.
The following example in Figure 4 shows a multiplex combination of assays by first recording the fluorescence resulting from the DNA binding dye to indicate the number of dead cells, followed by subsequent addition of a luminogenic reagent to quantify ATP as a marker for the number of viable cells. The multiplex combination of orthogonal assays to detect complementary cell health markers can be used to confirm overall results.
Controls were cultured in zeolite free media were run in parallel to the treatment group. Ferrous sulfate FeSO 4 was used as positive control.
Figure Lengend Snippet: P. A Representative gross picture of P. Typical DAD, hyaline membranes and bleeding were induced by Pa 4 exoproducts. B Histological difference of mice died with pneumothorax or not 3 h after high concentration of exoproducts instillation. F Gelatin zymography of exoproducts. G Fibrinogen degradation assay of exoproducts. H Thrombin degradation assay of exoproducts.
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