• Cytotoxicity Assay (MTT)

Cytotoxicity Assay (MTT)

A. Reagents and Preparation

Reagents

• MTT Solution: Dissolve 500 mg MTT powder in 10 ml PBS. Stir with a magnetic stirrer for approx. 1 h in the dark. Filter the solution with a 0.22 mm filter, and store in 10 ml aliquots at −20 °C in the dark.
• Acidified Isopropanol –Add 50 ml 2 M HCl to 2.5 l Isopropanol. Store the solution for at least 1 month at room temperature before use. This solution is used to dissolve the formazan crystals. Other solutions such as methanol, ethanol, and DMSO are also suitable. Note: If the isopropanol is not correctly acidified, the suspension will become cloudy. 

Equipment

• 96 well flat-bottomed microplates
• Benchtop centrifuge
• Microtiter plate reader (e.g. with 650- and 570-nm filters)
• Single-channel pipettes (0.001 – 1 ml) 
• Multi-channel pipettes (0.01 – 0.3 ml) 
• 37°C incubator 
• Sterile pipette tips
• Laminar flow hood (e.g. class 2B)

Plate Set-up

1. Each plate should contain control wells (without drugs) and blank wells (without cells).
2. An additional control is required of wells with medium (without cells) including the range of the drug if the drug shows absorbance at the given wavelengths.
3. Plate samples, controls and blanks as duplicates or triplicates, to minimise result variability.
4. A standard MTT assay requires: a testing plate to measure the drug OD, a testing plate to determine the growth curve for the starting amount of cells seeded per well, and a broad dilution range to determine the dilution range for the experiments. Note: For a standardised MTT drug sensitivity assay using patient samples, a single plate often suffices.
5. For cell lines, it is also recommended to include a day 0 plate for determining the extent of cell growth throughout the experiment.
6. Do not use the outer wells of the plate (due to evaporation). Fill the outer wells with PBS to minimise plate evaporation.
7. Some drugs can also have influence on neighbouring wells - this should be investigated before starting the experiment.

B. Procedure

If the stability of the drug is unknown, the plates should be freshly prepared following steps 1-3:

1. Add 30 µl of the drug concentration/dilution stock to each well, excluding control wells.
2. Add ~120 µl cells (1000-100000 cells) into each well, except blanks. The optimal concentration of cells used is dependent on the cell line/type.
3. The total volume of the cell and drug suspension should be 150 µl for each well, excluding blanks and controls. Note: For stable drugs which can be stored at −20 °C, the plates can be prepared with the 30 µl drug concentrations and stored at −20°C for later use. Cells can be added and mixed with the drug suspension when the assay is run. 
4. Incubate the plates for 72-96 hours with 5% CO2 at 37 °C, depending on the cell line/type used. 
5. After the appropriate incubation time, add 5 mg/mL (1:10 volume) of MTT solution to each well. Unused MTT can be frozen at −20 °C and reused. 
6. Shake plates for 5 min on a plate shaker by slowly increasing the shaking speed to a maximum of 900 shakes per minute. 
7. Incubate the plate for 4-6 hours at 37°C in a CO2 incubator. Note: The incubation time is dependent on the cell type.
8. Add 150 µl acidified isopropanol (or alternative reagent) to each well, and re-suspend until all Formazan crystals have been dissolved. 
9. Mix each well thoroughly using a multi-channel pipette. Between each row, rinse tips of multi-channel pipette with isopropanol and discard the used isopropanol. Blow out tips thoroughly before mixing the next rows. Start with the control wells, before mixing the rows with drugs. 
10. Leave the plate to rest at room temperature in the dark for at least 10 minutes before recording absorbance measurements.

Optical Density (OD) Measurement

The OD is measured at 540 and 720 nm to gain a more exact measurement by correcting for background noise. Subtract the 720 nm OD background from the 540 nm OD total signal. To calculate the percentage of living cells, use the following formulae:

• Control wells (cells, no drug): (Average OD of control wells) − (Average OD of blank wells)
• Drug wells (containing cells): (Average OD of drug wells) − (Average OD of blank wells)
• If the drug interferes with the OD measurement: (Average OD of control wells) − (Average OD of drug wells (drug, no cells)).
• The cell survival is then calculated by: (OD treated well [−blank]) / (mean OD control well [−blank]) × 100. The LC50, or the drug concentration which results in 50% cell survival, can then be calculated.