MTT Cell Proliferation And Cytotoxicity Assay Kit

The MTT method, also referred to as the MTT colorimetric method, is utilized to detect cell growth and survival. This technique relies on the principle that living cells possess succinate dehydrogenase, an enzyme found in their mitochondria, which can convert MTT into a water-insoluble blue-purple crystal called formazan. This formazan accumulates within the cells, while non-viable cells lack this ability. The amount of formazan produced is directly proportional to the number of cells within a specific range. A higher absorbance indicates increased cell activity, indicating lower toxicity of drugs. This highly sensitive and cost-effective method finds extensive application in assessing the efficacy of biologically active substances, large-scale anti-tumor drug screenings, cytotoxicity evaluations, and determining tumor radiosensitivity. The necessary components for this method include MTT (25mg), MTT solvent (5ml), and Formazan solution (55ml).

1. Start by placing the cell plate on a sterile operating table ensuring a clean environment for the experiment.
2. Proceed by adding a 10% volume of MTT solvent to each well of the cell plate.
3. To prepare the MTT solution, dissolve 25mg of MTT in 5ml of MTT solvent to create a 5mg/ml MTT solution. This solution can be used immediately or stored at -20°C, protected from light. If needed, it can be divided into smaller portions and stored at -20°C.
4. Next, return the cell plate to the incubator and allow it to incubate for approximately 3-4 hours. It is important to ensure consistent incubation time when conducting comparison experiments.
5. Once the incubation is complete, carefully remove the cell plate from the incubator. If working with adherent cells, discard the culture medium and add an equal volume of Formazan Solvent. While the volumes in each well might vary, it is crucial to maintain the same total volume for comparison purposes. However, if removing non-adherent cells or the culture medium risks the loss of MTT formazan, simply add an equal amount of dissolving solution to the initial volume of the culture medium.
6. After adding the Formazan Solvent, it is imperative to complete the reading within 1 hour. Gentle shaking using a shaker or repeated pipetting may be necessary to ensure complete dissolution of the MTT formazan crystals, especially if the cell density is high.
7. Utilize a spectrophotometer to measure the absorbance at OD570 and the reference wavelength OD690.
8. If using a 96-well plate, you can conveniently test it on a microplate reader equipped with appropriate filters. However, for other types of multiwell plates, a suitable size cuvette or plate reader is required for precise spectrophotometric detection.
Ensure to follow these steps precisely to achieve accurate and reliable results in your experiment.

Evaporation can be a problem when using the 96-well plate for testing, especially if the cell culture time is prolonged. To combat this, two strategies can be implemented. The first is to discard the outer circle of wells and replace with either PBS, water, or culture medium. The second is to place the 96-well plate near a water source to minimize evaporation. If the test compound reacts with MTT, the culture solution can be discarded after centrifugation and rinsed with PBS 2-3 times. The culture solution containing MTT can then be added. While Formazan produced is proportional to the number of living cells, it's critical to note that the duration of action also plays a role. Therefore, for samples with more specimens, the result of determination may change over time. Since this product contains carcinogens, please handle it with caution, taking care not to come into contact with skin or eyes. Wear lab coats and disposable gloves for your safety and health. Lastly, remember that this product is solely for scientific research purposes and isn't intended for clinical use or diagnosis.

Store at 2~8° C away from light for 12 months.

MTT Cell Proliferation and Cytotoxicity Assay Kit;MTT kit