Flash link: https://vlab.amrita.edu/repo/BIOTECH/CEL/Cell_Proliferation/index.swf

 

Materials Required

·    Micro titer plate (flat-bottomed)
•    Sterile   tube (5 ml)
•    Multi-channel reagent boat
•    Sterile pipette tips
•     Micropipettes
•    Multi-channel pipette
•    Beaker
•    Aluminum foil
•    Microtiter plate reader (with 650- and 570-nm filters)
•    CO2 incubator- 37°C
•    Vortex apparatus
•    Laminar Air flow

Reagents

•    70% Ethanol
•    MTT  reagent (5mg/ml)   -  5ml
•    Unknown drug
•    αMEM -50 ml
•    DMSO – 10ml
•    MTT solvent (  4mM  HCl , 0.1% Nondent P- 40(NP40) prepared in isopropanol)-  5ml

 

Reagent Preparation

Drug concentration

First stock concentration         - 10 µM in DMSO
Second stock concentration    -100 µM in DMSO

Dilution of first stock concentration (10 µM).

1.    200nM :  980 µl of DMEM is added with 20 µl  of first drug  concentration.
2.    400nM :  960 µl of DMEM is added with 40 µl  of first drug  concentration.
3.    600nM :  940 µl of DMEM is added with 60 µl  of first drug  concentration.
4.    800nM :  920 µl of DMEM is added with 80 µl  of first drug  concentration.

Dilution of second stock concentration (100 µM).

1.     1µM :     990 µl of DMEM is added with 10 µl  of second drug  concentration .
2.    1.5 µM :  9805µl of DMEM is added with 15 µl  of second drug  concentration.
3.    2 µM :     980 µl of DMEM is added with 20µl  of second drug  concentration.
4.    2.5 µM :  975 µl of DMEM is added with 25 µl  of second drug  concentration.

 

Procedure

1.    The cells were plated at a density of 5,000- 10,000 per well ad incubated overnight.


2.    Remove cultures from the incubator into the laminar flow hood.


3.    Cells were treated with the unknown compound at different concentrations ( e.g. 200nM, 400nM, 600nM, 800nM, 1µM,   1.5µM, 2µM, 2.5µM) for 24 hours. [control- untreated, blank –medium alone.The experiment was done more than triplicates] using pipettes.


4.    Add 20µl of 5mg/ml of MTT  reagent is added to each well and incubated for 3  hours. At 37°C in the incubator.


5.    The medium is removed and add 150µl of MTT solvent  was added to each well.


6.    Gently pipetting up and down may be required to completely dissolve MTT formazan crystal.


7.    The plate was covered with aluminium foil and kept at room temperature for few minutes.


8.    Measure the absorbance of multi well plate at 570nm, and 620nm.