I am doing an MTT assay to determine whether my natural compound (let's call it X) has cytotoxic effects on the cell-line I am using.
The natural compound has been extracted and semi-purified. In order to determine the actual content of the compound in this extract, I have measured the absorbance using spectrophotometry in which I then applied Beer-Lambert Law and obtained a value of 60.8 mg/g.
The liquid I will be using to dissolve my extract in is Milli-Q water. Using a 2-fold dilution, I will then prepare the different concentrations, and to do this, I will be using 1 X complete MEM as the diluent.
I will use 8 different concentrations for the MTT assay (4 replicates of each concentration):
2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, 0.03125 mg/mL, and 0.015625 mg/mL
To obtain these concentrations, how do I prepare my extract? How do I know how much of the extract is to be dissolved in water, and thereafter how much of it needs to be mixed with 1 X MEM when giving the treatment?
Thank you so much in advance!
First you need to prepare a mother dissolution, this is, a dissolution of your compound that has a higher concentration than all the final dissolutions you need, and enough volume to prepare them all. Then you take aliquotes from that dissolution and prepare the child (final) dissolutions.
Your mother dissolution could be 2 mg/mL for instance, as this is the highest concentration you need. To calculate how much you need you need to do the calculations of how many aliquotes of which volumes you will take from it, and this depends on the volume you need of the final dissolutions, which you haven't stated. You calculate this using the equation C1·V1=C2·V2, where C1 and V1 are the concentration and volume of the mother dissolution (the aliquote you will take from the 2 mg/mL dissolution, this will be C1) and C2 and V2 are the concentration and volume of the final dissolutions (the concentrations you have stated, volumes are missing).
For preparing the mother dissolution (100 mL of 2 mg/mL, for example), you will need to take into account the purity:
2 mg/mL · 100 mL / 60.8 mg/g = 3.2895 g would be the ammount of unpure solid you need to weight and dissolve in 100 mL of Milli-Q water.
In your case you need a serial dilution. This is that in each step that you dilute you will be reducing the concentration by half. You need to prepare a solution of your extract at a concentration of 4 mg/ml. Once in the well, half the volume is taken and transferred to the other well, and so on until the last well is discarded.
Greetings
