Cell Cytotoxicity Assay

Membrane integrity is the most commonly used characteristic for detecting cell death in vitro culture. Inactive or dead cells will lose membrane integrity and allow other impermeable molecules to enter. Dead cells can be detected by measuring the movement of molecules passing through the cell membrane that has already leaked and cannot be repaired, entering and exiting the cell. The principle of MTT detection is that succinate dehydrogenase in live cell mitochondria can reduce exogenous MTT to water-soluble blue purple crystalline Formazan and deposit it in cells, while dead cells do not have this function. Dimethyl Sulfoxide (DMSO) can dissolve Formazan in cells, and its light absorption value is measured at a wavelength of 490nm, which can indirectly reflect the number of live cells. In vitro cytotoxicity testing has become a component of drug discovery as it is a convenient, cost-effective, and predictable method for characterizing the potential toxicity of new chemical entities.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Cell Culture

Due to the different sources of cells, the methods of cell incubation also vary. For specific protocols, please refer to the cell culture topic on our website.

2. MTT Assay for Evaluating Cytotoxicity

2.1 MTT solution

• Weigh 0.5 g of MTT and dissolve it in 100 mL of PBS for clarification, with a concentration of 5 mg/mL.

• Filter and sterilize the MTT solution using a 0.2 µm filter into a sterile, dark container.

3. MTT dissolution solution

3.1 DMSO can be directly dissolved.

3.2 Solubilization Solution:

• Choose a suitable solvent resistant container and work in a fume hood. Prepare 40% (volume/volume) Dimethylformamide (DMF) in 2% (volume/volume) Acetic Acid.

• Add 16% (weight/volume) Sodium Dodecyl Sulfate (SDS) and dissolve.

• Adjust to pH=4.7.

• Store at room temperature to avoid SDS precipitation. If the precipitation is formed, you can heat to 37°C and mix to dissolve SDS.

4. MTT testing

4.1 Add cells and test compounds to a 96 well plate, with a final volume of 100 µ L/well. Incubate for the required exposure time (specific process can be found in cell culture).

4.2 Add 10 µ L of MTT solution to each well to achieve a final concentration of 0.45 mg/ml. Incubate at 37 ° C for 1-4 hours, discard the supernatant, and centrifuge the suspended cells first.

4.3 Add 100 µ l of DMSO or a dissolved solution to each well and shake for 10 minutes to fully dissolve the formate crystals, avoiding the formation of bubbles.

4.4 Measure the absorbance at 490 nm or 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader or spectrophotometer.

5. Calculation formula

Cell survival rate=[(As-Ab)/(Ac-Ab)] × 100%

Inhibition rate=[(Ac-As)/(Ac-Ab)] × 100%

As: absorbance of experimental wells (including cells, culture medium, MTT solution, and drug solution);

Ac: absorbance of control wells (including cells, culture medium, MTT solution, excluding drugs);

Blank well absorbance (including culture medium and MTT solution, excluding cells and drugs).

1. Cell density and drug concentration have a significant impact on the experimental results. Based on experience, it is recommended to have around 4000 cells per well when laying the board, and to control the cell density at around 30% during administration. The cells at the bottom of the hole must be evenly distributed.

2. When using a 96 well plate for testing, if the cell culture time is long, be sure to pay attention to the issue of evaporation.

3. MTT needs to be stored in dark. During the experiment, it is recommended to operate quickly and temporarily do not need to be stored in dark.

4. Avoid compounds containing Thiols in the solution, as they can convert MTT into Formazan, resulting in false positive data.

5. The presence of Ahenol Red in the culture medium may also affect the experimental results.

6. Before adding DMSO, the liquid should be sucked cleaning, otherwise the precipitate will be difficult to dissolve.