1. Make solution of 5 mg/ ml of MTT dissolved in PBS and fikter sterilise

2. 5 hours before the end of incubation add 20 micro l of MTT solution of step one to each well containing cells.

3. Incubate the plate ( 96 well plate ) , in a CO2 incubator at 37 degree Cepcius for 5 hours

4. remove media with needle n syringe

5. add 200 micro l of DMSO tobeach well an pipette up and down to dissolve crystal

6. Place plate into 37,5 degree C incubator for 5 minutes

7. Transfer to plate reader and measure adsorbance at 550 nm

Step 1: Determine the Desired Stock Concentration

Step 2: Calculate the Amount of 2,4-DTBP for the Stock Solution

• The molecular weight of 2,4-DTBP = 206.33 g/mol.
• To make a 10 mM stock solution in DMSO: calculate

Step 3: Prepare Working Solutions for the MTT Assay

Decide on the final concentrations of 2,4-DTBP you want to test on MCF-7 cells in the MTT assay, e.g., 0.1 µM, 1 µM, 10 µM, and 50 µM.

Example of Serial Dilutions as follows

Let’s go step-by-step to make working solutions from the 10 mM stock:

These working solutions allow you to add small amounts to your cell culture medium to achieve the final concentrations needed for the MTT assay.

Step 4: Calculate Final Concentrations in Cell Culture Medium

2 to 3 hours is enough for MTT incubation based on my experience