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
5.
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.
8.
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.