Cell Viability and Proliferation
By: Mark Frei, BioFiles v6 n5, 17–21
BioFiles Volume 6, Number 5 — Centrifugation
Download BioFiles v6 n5 (4.66 Mb PDF)
Assays to measure proliferation, viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) is a popular choice for measuring the number of cellular divisions a population has undergone. Upon entering the cell, CFSE is cleaved by intracellular esterases to form the fluorescent compound and the succinimidyl ester group covalently reacts with primary amines on intracellular proteins. Upon division, the fluorescence intensity of each daughter cell is halved which allows for the simple detection of the number of cell divisions by flow cytometry.
Assays that measure metabolic activity are suitable for analyzing proliferation, viability, and cytotoxicity. The reduction of tetrazolium salts such as MTT and XTT to colored formazan compounds or the bioreduction of resazurin only occurs in metabolically active cells. Actively proliferating cells increase their metabolic activity while cells exposed to toxins will have decreased activity.
Cell Viability Applications
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue) is a water soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes.1 This water insoluble formazan can be solubilized using isopropanol or other solvents and the dissolved material is measured spectrophotometrically yielding absorbance as a function of concentration of converted dye.
The cleavage and conversion of the soluble yellow dye to the insoluble purple formazan has been used to develop an assay system alternative to the conventional 3H-thymidine uptake and other assays for measurement of cell proliferation. Active mitochondrial dehydrogenases of living cells will cause this conversion. Dead cells do not cause this change. This has been applied in measurement of interleukin-2 activity in a multiwell assay.2 Modification has improved the sensitivity.3 Other uses such as measurement of cytotoxicity4 and cell number have also been developed.
In our testing we dissolve MTT, (Cat. No. M5655), 5 mg/ml in RPMI-1640 without phenol red. This medium is available as a powder (Cat. No. R8755) or liquid (Cat. No. R7509). The solution is filtered through a 0.2 μm filter and stored at 2–8 °C for frequent use or frozen for extended periods.
Routinely, MTT stock solution (5 mg/ml) is added to each culture being assayed to equal one-tenth the original culture volume and incubated for 3 to 4 hr. At the end of the incubation period the medium can be removed if working with attached cells and the converted dye may be solubilized with acidic isopropanol (0.04-0.1 N HCl in absolute isopropanol). When working with suspension cells the dye is added directly and dissolution is accomplished by trituration. Absorbance of converted dye is measured at a wavelength of 570 nm with background subtraction at 630–690 nm.
MTT may also be used to score hybridoma development or clonal development. Clones will convert the dye and become readily visible without magnification. It should be noted that MTT is a mutagen and the resultant cells may be affected. The concentration used may be reduced by dilution of the stock solution (5 mg/ml) to 0.1 mg/ml and adding a tenth volume to each well. Incubation should be monitored by observing for stained clones. Cells can be recovered by gently washing the cells and adding growth medium.
MTT stock solution: 5 mg/mL
Typical use: Add 1/10th of culture volume
Solvent: 0.04-0.1 N HCl in isopropanol
Can also use DMSO:isopropanol (1:1)
Spectrophometric reading: 570 nm
Background wavelength: 630-690 nm
Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the visualization of cell morphology.
NOTE: Trypan Blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting.
Protocol for Viable Cell Counting using Trypan Blue
- Prepare a cell suspension in a balanced salt solution (e.g., Hanks' Balanced Salts [HBSS], Cat. No. H9269).
- Transfer 0.5 ml of 0.4% Trypan Blue solution (w/v) to a test tube. Add 0.3 ml of HBSS and 0.2 ml of the cell suspension (dilution factor = 5) and mix thoroughly. Allow to stand for 5 to 15 minutes.
Note: If cells are exposed to Trypan Blue for extended periods of time, viable cells, as well as non-viable cells, may begin to take up dye.
- With the cover-slip in place, use a Pasteur pipette or other suitable device to transfer a small amount of Trypan Blue-cell suspension mixture to both chambers of the hemacytometer. Carefully touch the edge of the cover-slip with the pipette tip and allow each chamber to fill by capillary action. Do not overfill or underfill the chambers.
- Starting with chamber 1 of the hemacytometer, count all the cells in the 1 mm center square and four 1 mm corner squares (see Diagram I). Non-viable cells will stain blue. Keep a separate count of viable and non-viable cells.
Note: Count cells on top and left touching middle line of the perimeter of each square. Do not count cells touching the middle line at bottom and right sides (see Diagram II).
- Repeat this procedure for chamber 2.
Note: If greater than 10% of the cells appear clustered, repeat entire procedure making sure the cells are dispersed by vigorous pipetting in the original cell suspension as well as the Trypan Blue-cell suspension mixture. If less than 200 or greater than 500 cells (i.e., 20–50 cells/square) are observed in the 10 squares, repeat the procedure adjusting to an appropriate dilution factor.
- Withdraw a second sample and repeat count procedure to ensure accuracy.
Cell Counts – Each square of the hemacytometer, with cover-slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equivalent to approximately 1 ml, the subsequent cell concentration per ml (and the total number of cells) will be determined using the following calculations:
Cells Per mL = the average count per square × dilution factor × 104 (count 10 squares)
Example: If the average count per square is 45 cells × 5 × 104 = 2.25 × 106 cells/ml.
Total Cells = cells per ml × the original volume of fluid from which cell sample was removed.
Example: 2.25 × 106 (cells/ml) × 10 ml (original volume) = 2.25 × 107 total cells.
Cell Viability (%) = total viable cells (unstained) ÷ total cells (stained and unstained) × 100.
Example: If the average count per square of unstained (viable) cells is 37.5, the total viable cells = [37.5 × 5 × 104] viable cells/ml × 10 ml (original volume) = 1.875 × 107 viable cells. Cell viability (%) = 1.875 × 107 (viable cells) ÷ 2.25 × 107 (total cells) × 100 = 83% viability.