The MTT Cell Proliferation Assay

INTRODUCTION

Measurement of cell viability and proliferation forms the basis for numerous in vitro assays of a cell population’s response to external factors. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means.

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields low background absorbance values in the absence of cells. For each cell type the linear relationship between cell number and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell proliferation.

Component Volume Storage

MTT Reagent 25 ml 4°C
Detergent Reagent 2 × 125 ml Room Temp. or 4°C

EQUIPMENT REQUIRED BUT NOT SUPPLIED

Microtiter plate reader with 650- and 570-nm filters
Inverted microscope
Multi-channel pipette
37°C incubator
Laminar flow hood
Microtiter plate (flat-bottomed)
Sterile tubes (5 ml)
Serological pipettes
Sterile pipette tips

BASIC PROTOCOL

If you are familiar with the procedure and know the cell count to use in your specific assay, you may follow this basic protocol.

1 、Plate cells at 1000 to 100,000 per well.

2 、Incubate for 6 to 24 hours

3 、Add 10 μl MTT Reagent

4 、Incubate for 2 to 4 hours until purple precipitate is visible.

5 、Add 100 μl Detergent Reagent.

6 、Leave at room temperature in the dark for 2 hours.

7、 Record absorbance at 570 nm.

Step Action

1、DETERMINING OPTIMAL CELL COUNTS

Use the protocol below to determine the optimal cell count and incubation period for your cell line.
This determination should only have to be done once for each cell type. The data will be used thereafter in your experimental system following the protocol above.

（1） Harvest suspension cells by centrifugation. Adherent cells should be released from their substrate by trypsinization or scraping.

（2 ）Resuspend cells at 1 x 106 per ml.

（3） Prepare serial dilutions of cells in culture medium from 1 x 106 to 1 x 103 cells per ml.

（4 ）Plate out, in triplicate, 100 μl of the dilutions into wells of a microtiter plate.

（5 ）Include three control wells of medium alone to provide the lanks for absorbance readings.

（6 ）Incubate the cells under conditions appropriate for the cell line for 6 to 48 hours (to recover from handling). The time required will vary but 12 hours to overnight is sufficient for most cell types.

（7 ）Add 10 μl of MTT Reagent to each well, including controls.

（8 ）Return plate to cell culture incubator for 2 to 4 hours.

（9 ）Periodically view the cells under an inverted microscope for presence of intracellular punctate purple precipitate.

（10 ）When the purple precipitate is clearly visible under the microscope add 100 μl of Detergent Reagent to all wells, including controls. Swirl gently; do not shake.

（11） Leave plate with cover in the dark for 2 to 4 hours or overnight at room temperature.

（12） Remove plate cover and measure the absorbance in each well, including the blanks, at 570 nm in a microtiter plate reader. [Absorbances can be read with any filter in the wavelength range of 550 - 600 nm. The reference wavelength should be higher than 650 nm. The blanks should give values close to zero (+/- 0.1).]

（13） If the readings are low return the plate to the dark for longer incubation.

（14） Determine the average values from triplicate readings and subtract the average value for the blank. Plot absorbance against number of cells/ml. The number of cells to use in your assay should lie within the linear portion of the plot and yield an absorbance of 0.75 - 1.25.

2、PERFORMING AN ASSAY

The plot of the data obtained in Step 14 (absorbance against number of cells) should provide a curve with a linear portion. The optimal number of cells for the assay should fall within the linear portion of the curve and give an absorbance value between 0.75 and 1.25.Then both stimulation and inhibition of cell proliferation can be measured. To run an assay, select an optimal cell number and follow the MTT Cell Proliferation Assay steps 4 to 13 using your experimental system, plating in triplicate. Assays will include:

（1）Blank wells containing medium only.

（2) Untreated control cells.

（3) Test cells treated with the substance to be assayed.

If more than 100 μl of medium is used per well, increase the amount of MTT Reagent accordingly;e.g., for 250 μl of medium use 25 μl of MTT Reagent.

DATA INTERPRETATION

Absorbance values that are lower than the control cells indicate a reduction in the rate of cell proliferation. Conversely a higher absorbance rate indicates an increase in cell proliferation.Rarely, an increase in proliferation may be offset by cell death; evidence of cell death may be inferred from morphological changes.

TROUBLESHOOTING

1、
Problem:
MTT Reagent is blue-green.

Cause
Contamination with a reducing agent or cell/bacterial contamination.
Excessive exposure to light.

Remedy
Discard. Remove aliquots of new MTT Reagent using sterile technique.
Store solution in the dark at 4°C.

2、
Problem:
Blanks (medium only) give high absorbance readings.

Cause
The medium is contaminated with cells/bacteria/yeast (visible under microscope).

Remedy
Discard. Check medium before plating. Use sterile technique for cell plating in biological hood. Use sterile 96-well plate.
The medium contains ascorbic acid. Incubate plate in the dark. Find alternative medium if possible.

3、
Problem:
Absorbance readings too high.

Cause
Cell number per well too high. Decrease cell density at plating.
Contamination of culture with bacteria or yeast.

Remedy
Discard. View wells prior to addition of MTT Reagent to check for contamination.

4、
Problem:
Absorbance readings are too low.

Cause
Cell number per well is too low.
Incubation time for reduction of MTT intracellularly too short. No purple color visible in cells when viewed under microscope.
Cells not proliferating due to improper culture conditions or inadequate time of recovery after plating.

Remedy
increase cell density at plating.
Increase incubation time with MTT Reagent until purple color is evident inside cells when viewed under microscope.
Longer incubation of up to 24 hours may be required for some cell types.
Incubation time for solubilization of formazan dye too short (intact cells with intracellular dye visible when viewed under the microscope).
Increase incubation time with Detergent Reagent or incubate at 37°C. View under microscope to ensure no crystals remain out of solution.
Check that culture conditions (medium, temperature, humidity, CO2, etc.) are appropriate. View cells periodically to check condition. Increase time in culture after plating for cell recovery.

5、
Problem:
Replicates have different values.

Cause ：
Inaccurate plating or pipetting.

Remedy：
Increase accuracy of cell plating, check accuracy of pipette.

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