This document describes the MTT assay, a colorimetric assay used to measure cell viability and cytotoxicity. The MTT assay works by using the enzyme mitochondrial dehydrogenase in living cells to reduce the yellow tetrazolium dye MTT to purple insoluble formazan. The amount of formazan produced is directly proportional to the number of viable cells. The document outlines the principle, reagents, procedure, troubleshooting, advantages, and disadvantages of the MTT assay. Commonly available MTT assay kits are also listed.

1. BY:
Syeda Sakeena Gilani
Mphil Leading to PhD
Microbiology

2. MTT Assay
 Colorimetric assay
 Sensitive
 Quantitative
 Reliable
 Purposes:
 For assessing cell viability
 To measure cytotoxicity (loss of viable cells)
 Cytostatic activity (shift from proliferation to
quiescence)

3. Principle
 Water soluble yellow MTT
 Reduced to purple insoluble formazan by mitochondrial
dehydrogenases
 Water insoluble formazan can be solubilized using
isopropanol or other solvents
 The dissolved material is measured spectrophotometrically
yielding absorbance as a function of concentration of
converted dye.

4. Contt.
 Presence of viable cells is detected as active
mitochondrial dehydrogenase of living cells will
cause this conversion.
 Non-viable cells will not produce dehydrogenase.
Thus, dead cells do not cause this change
 The amount of formazan produce is directly
proportional to the number of viable cells in the
sample

6. Reagents Preparation
 MTT solution:
 5mg/ml MTT in PBS.
 Solution must be filter sterilized (0.22um filter) after
adding MTT.
 For long term storage keep the MTT solution in
-20°C.
 MTT solvent:
 Option1: 400ul HCl 1M, 10ml triton 100%, 90ml
isopropanol.
 Option2: DMSO 100% room temperature.

7. Procedure
Plate cells at 1,000-100,000
per well.
Incubate for 6-24 hours.
Add 10μL MTT Reagent.
Incubate for 2-4 hours until
purple precipitate is visible.
Add 100μL Detergent
Reagent.
Leave at room temperature
in the dark for 2 hours.
Record absorbance at
570nm.

9. Trouble Shooting
 Problems
 MTT Reagent is
blue-green.
 Blanks give high
absorbance
readings
 absorbance
readings too high
 Remedies
 Discard it and Store
solution in the dark
at 4°C.
 May be due to
contamination. Use
aseptic techniques.
 Decrease cell
density at plating.

10.  Replicates have
different values.
 Absorbance readings
are too low.
 Increase accuracy of cell
plating, check accuracy of
pipette.
 Increase cell density at
plating.
 Increase incubation time with
MTT reagent or with
detergent reagent
 Check that culture conditions
are appropriate. View cells
periodically to check
condition.
 Increase time in culture after
plating for cell recovery

11. Advantages
 No transfer of the cells; the entire assay is
performed in a single microplate.
 MTT is metabolized by all cells; the assay
can be used with all cell types.
 Inexpensive

12. Disadvantages
 Assay is not linear over a broad logarithmic
cell proliferation range due to the ELISA plate
reader.
 Insoluble reaction product; resolubilization of
the reaction product required.
 Cannot take multiple time points in a single
assay.
 Cells with low metabolic activity (e.g.,
lymphocytes) must be used in high numbers.

13. Commercially Available Kits
 CellTiter 96® Non-Radioactive Cell Proliferation
Assay. Promega Corporation Cat.# G4000
 Cell Growth Determination Kit, MTT based.
Sigma Aldrich Cat.# CGD1-1KT
 MTT Cell Growth Assay Kit. Millipore Cat.#
CT02.
 Thiazolyl Blue Tetrazolium Bromide (MTT
Powder). Sigma-Aldrich Cat.# M2128.