General: Definitions and Techniques
FAQ: How can I determine how fit my cells are?
HelpCorner: Usually, one of two parameters is used to determine the health of the cells: cell viability or cell proliferation.
Cell viability can be defined as the number of healthy cells in a sample. Cell viability assays are often useful when non-dividing cells (such as primary cells) are isolated and maintained in culture to determine optimal culture conditions for these populations. The most useful and straightforward method for determining viable cell number is to stain the cells with a dye such as trypan blue and count them in a hemocytometer (e.g., the Neubauer hemocytometer). The dye allows to distinguish between healthy cells with uncompromised membrane integrity (unlabeled) and non-healthy ones (colored blue). One can also measure metabolic activity by incubating cells with tetrazolium salts that are cleaved into colored, water-insoluble (MTT) or water-soluble (XTT, WST-1) formazan salts. WST-1 is stable enough to be packaged as a ready-to-use solution.
Cell proliferation is the determination of the number of dividing cells. One way to measure this parameter is to perform clonogenic assays. In these assays, a defined number of cells are plated onto an appropriate matrix, and the number of colonies formed after a period of growth are counted. Drawbacks to this type of technique are that it is tedious and not practical for large numbers of samples. Another way to analyze cell proliferation is to measure DNA synthesis. In these assays, labeled DNA precursors (3H-thymidine or bromodeoxyuridine, BrdU) are added to cells, and their incorporation into DNA is quantified after incubation. The amount of labeled precursor incorporated into DNA is quantified either by measuring the total amount of labeled DNA in a population, or by detecting the labeled nuclei microscopically. Cell proliferation can also be measured using indirect parameters. In these techniques, molecules that regulate the cell cycle (also called proliferation markers) are measured either by their activity (e.g., CDK kinase assays) or by quantifying their amounts (e.g., Western blots, ELISA, or immunohistochemistry).
Methods for Studying Cell Proliferation and Viability
FAQ: Which product do you recommend for quantifying cell viability?
HelpCorner: We recommend the use of Cell Proliferation Kit I (MTT) or II (XTT) or Cell Proliferation Reagent WST-1. All assays can be performed on adherent or suspension cells. Please note that only MTT is metabolized by all cells. For multiple time points, use either XTT or WST-1 tetrazolium salts.
FAQ: Is it possible to simultaneously measure cell viability and proliferation?
HelpCorner: An example of the simultaneous use of Cell Proliferation ELISA BrdU and Cell Proliferation reagent WST-1 for examining DNA synthesis and metabolic activity is provided in the Apoptosis and Cell Proliferation Manual, 3rd edition, p88 and in Biochemica 3/2003 (Figure 1).
FAQ: Which kit should I use for detecting BrdU-labeled DNA in proliferating individual cells?
HelpCorner: We recommend using BrdU Labeling and Detection Kits I or II. Both assays can be performed with cultured or freshly dissociated cells.
FAQ: Is it possible to combine the BrdU Labeling Detection Kit I and another antibody in order to double-stain the cells? If possible, which step should be performed first?
HelpCorner: To perform double-staining, first follow the protocol of the BrdU Labeling Detection Kit up to the fixation step. Then add the second primary antibody. After washing, add the anti-BrdU antibody as indicated in the pack insert, followed by the secondary antibodies. In principle, this could be done in one step, but this depends on your second primary antibody.
FAQ: Which product should I use for quantifying DNA synthesis during cell activation and proliferation?
HelpCorner: You should use the Cell Proliferation ELISA, BrdU (colorimetric or chemiluminescent). The principle of this assay is to incubate cells with BrdU and analyze the amount of labeled incorporated BrdU by immunodetection. The assay can be performed on adherent or suspension cell cultures.
FAQ: Regarding the Cell Proliferation ELISA, BrdU (colorimetric), how important is the reference wavelength of 690 nm? There are two options with the dual filter reader: subtract or ratio. Which one should I use?
HelpCorner: The exact value for the reference wavelength is not critical, but it must be higher than 620 nm. You should subtract the reference absorbance from the test absorbance measured at 450 nm.
FAQ: What is the proper formula for calculating the stimulation index using the Cell Proliferation, BrdU?
HelpCorner: You should first calculate the average of triplicates and subtract the background value (without cells) of each sample (stimulated cells) and negative control (unstimulated cells). Then the values of samples are divided by the negative control. The data obtained are relative values which can be compared between different tests.
FAQ: Using the Cell Proliferation ELISA, BrdU on stimulated human lymphocytes, we obtained very low values. What could be the reason for this low efficiency?
HelpCorner: To study lymphocyte proliferation, cells are stimulated (e.g., with growth factors, cytokines or mitogens). The increase in cell numbers can (in special cases) lead to cluster formation of the lymphocytes: Cells from the same progenitor stick together and form aggregates in the culture plate. This effect may disturb the antibody recognition of the ELISA system and thereby result in an underestimation of the response. To avoid signal variation, carefully resuspend the cells by pipetting after the BrdU-labeling period and prior to removing the culture medium. This will enable equal accessibility of each cell for antibody recognition of the BrdU-label.
This problem mostly affects suspension cells such as lymphocytes. Should you experience this problem with adherent cells, remove the labeling medium after the labeling period is completed, carefully trypsinize the cells to generate single cells, centrifuge the microplate and dry the cells as described in the pack insert for suspension cells. Proceed with the standard procedure.
FAQ: How can I reduce the background when I am using the BrdU Labeling and Detection Kit II ?
HelpCorner: Most of the time, the background can be due to the plastic of your plates or to an incomplete washing step. The anti-BrdU-POD antibody may bind unspecifically to some plastic material. Always use sterile flat-bottomed microplates that are anti-adsorbent (e.g., Maxisorp from NUNC or Costar).You can also do a further blocking step with PBS/1–5% BSA before adding the antibody. In some cell lines, a high cell concentration is the origin of the unspecific binding of the antibody. In this case, decreasing the cell number will help to reduce the background.
1. Heath JR (2001) Principles of Cell Proliferation, Blackwell Publishing
2. Hughes D et al. (2002) Cell Proliferation and Apoptosis, BIOS Scientific Publication
3. Apoptosis and Cell Proliferation Manual, 3rd edition (https://www.roche-applied-science.com/sis/apoptosis/docs/manual_apoptosis.pdf)
This article was originally published in Biochemica 2/2008, pages 32-33. ©Springer Medizin Verlag 2008