MTT assay is used for detection of cellular viability ant cytotoxicity of chemicals

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.