MTS Reagen for Detecting Cell Viability
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Preparation of MTS Working Solution
(1) Preparation of Stock Solution: Weigh 42mg of MTS reagent powder and add to 21mL of DPBS. Mix on a magnetic stirrer at moderate speed for 15 minutes or until the MTS Reagen is completely dissolved. Measure the pH of the MTS solution. The optimal pH range is 6.0–6.5. If the pH exceeds 6.5, adjust to pH=6.5 using 1N HCl. Dissolve 38.3mg of phenazine methosulfate (PMS) in 10mL of DPBS. Sterilize the MTS Reagen solution by filtering through a 0.2μm membrane. Store the solution in the dark at -20°C.
(2) Preparation of Working Solution: Prepare 2.1mL of working solution by combining 2mL of MTS Reagen stock solution with 100μL of PMS solution. Mix gently by swirling or pipetting.
2. Cell Treatment and Staining
(1) Cell Seeding: Seed cells into a 96-well plate, typically adding 100μL of culture medium per well.
(2) Staining: Add 20μL of the MTS Reagen/PMS working solution to each well of the 96-well plate (containing 100μL of medium per well). Gently mix the plate to ensure uniform distribution.
(3) Incubation: Incubate the plate at 37°C in a humidified 5% CO₂ atmosphere for 1–4 hours, protected from light.
3. Signal Detection
(1) Immediate Measurement: After incubation, immediately measure the absorbance at 490nm using a microplate reader.
(2) Delayed Measurement: To delay measurement, add 25μL of 10% SDS to each well to terminate the reaction. Store the plate protected from light in a humidified chamber at room temperature for up to 18 hours before measurement. Note: The optimal detection wavelength is 490 nm, but measurements can be taken within 450–540nm. A reference wavelength of 630–700nm may be used to eliminate background interference.
Precautions:
(1) MTS reagent is light-sensitive. Perform all steps protected from light.
(2) Strong reducing agents (e.g., vitamin C, thiol-containing compounds) or high pH in the culture medium may increase background absorbance. Control experimental conditions carefully.
(3) PMS is a carcinogen. Dispose of waste according to safety regulations.
(4) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Martin F, Neubert A, Lutter AH, et al. MTS, WST-8, and ATP viability assays in 2D and 3D cultures: Comparison of methodologically different assays in primary human chondrocytes. Clin Hemorheol Microcirc. 2024;88(s1):S3-S19.
