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Protocol：MTT Assay

 

MTT Assay Protocol

1. Prepare MTT Solution (5 mg/mL in PBS)

·       - Filter sterilize the solution using a 0.22 μm filter.

·       - Aliquot and store at –20°C (stable for at least 6 months).

2. Discard Culture Media

·       - Adherent cells: Carefully aspirate the media.

·       - Suspension cells: Centrifuge the plate at 1,000 × g, 4°C, 5 min, then carefully aspirate media.

3. Add Reagents

·       - Add 50 µL serum-free media to each well.

·       - Add 50 µL MTT solution (5 mg/mL) to each well.

·       - If culture medium contains serum or phenol red, set up background control wells with 50 μL MTT solution + 50 μL media (no cells).

·        Incubate at 37°C for 3 hours.

·       - Add 150 µL of DMSO to each well.

4. Dissolve Formazan

·       - Wrap the plate in foil to protect from light.

·       - Shake on an orbital shaker for 15 minutes.

5. Measure Absorbance

·       - Read absorbance at OD = 590 nm.

6. Data Analysis

·       - Calculate mean OD for replicate wells.

·       - Subtract background absorbance (media + MTT, no cells) to get corrected absorbance.

·       % Cytotoxicity = 100 × (Control - Sample) / Control

 

 

By Xuehao

05/22/2025

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