INVITTOX Life

MTT test

Cells:

Any cells with recognisable levels of mitochondrial activity

Principle of MTT test:

After 3-4 hours of cell incubation with MTT formazan crystals develop in living and early apoptotic cells
Dead cells don’t produce formazan crystals

MTT
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide
TOXIC !
wear protection while handling

Basic materials:

HBSS

MTT

Solvent: DMSO+ethanol (1:1)

several other solvent combinations can be used

Basic equipment:

96 well plates

plate shaker

plate reader

Cell seeding:

Seed 100µl of cell suspension (usually 5x10⁴/ml) 96-well tissue culture plate
use six channels pipette
To avoid evaporation, use only central part of the plate (60 internal wells)

Exposure :general rules

Peripheral wells:If possible try to avoid using peripheral wells
Evaporation can considerably affect the cell growth in those wells
Fill the wells with medium only

Exposure :general rules
Two sets of controls
(untreated cells) on both sides of the plate.
Differences between left and right control indicate the influence of seeding,
humidity and/or temperature on the obtained results

Preparing MTT stock solution:

Dissolve MTT in HBSS to obtain final conc. 5 mg/ml
Sonicate solution
MTT solution can be stored at -20^oC

Adding MTT stock solution:

After incubating as required, add 10µl of MTT solution to each well

final MTT conc. 0.5 mg/ml

Incubation:

Incubate for 3-4 hours

Medium removal after incubation:

Invert plate rapidly to remove medium

Gently tap the plate on paper tissue to complete medium removal (violent shaking results in formazan loss and decreased accuracy)

Adding solvent:

Solubilise formazan crystals with 200 µl of organic solvent

Pay special attention to pipetting accuracy Every time check the level of liquid in pipetting tips

Solubilisation process:
Place the plates on a shaker and agitate for 10-20 minutes

Inspect the plate, as undissolved crystals affect the quality of your test

Do not stack the plates, as it results in non- uniform mixing

Plates after solubilisation of formazan crystals:

1 substance

2 substances

Formazan solution absorbs light at 550-570 nm but not at 620-650nm

620-650 nm absorbency results from cell debris & well imperfections

Final optical density (OD) obtained from formazan formation can be calculated OD=L1-L2

Reading:

Read the plates at 550-570 nm and 620-650nm as reference on scanning multiwell spectrophotometer

Record the optical density of each well

Percentage cell survival is expressed as:

(absorbance treated wells / absorbance of control wells) x 100%

Dose response curves can be calculated for chemicals tested over a range of concentrations, enabling IC50 values to be obtained (i.e. concentration of chemical resulting in 50% inhibition of cell growth or reducing cell survival by 50%)

Links to related INVITTOX protocols: