Materials Required

• Micro titter plate (flat-bottomed)

• Sterile tube (5 ml)

• Multi-channel reagent boat

• Sterile pipette tips

• Micropipettes

• Multi-channel pipette

• Beaker

• Aluminium 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.

 

Steps

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