I used 15:1 ratio to mix exosomes & Temozolomide drug (300 μL Exos + 20 μL TMZ).

My TMZ stock solution is 1 mg/ml, so 20μl = 20μg TMZ.

Then, I sonicated/incubated the mixture to load the TMZ into Exos. After that, I centrifuged the the mixture to separate free TMZ (supernatant) and TMZ-loaded Exos (pellet). Then, I put 200μL from 1 mL of supernatant into 96-well plate to determine the concentration of supernatant using indirect method formula. So, if the concentration that I get is 10 μg/ml, how to get the amount of weight of free TMZ? What does this 10 μg/ml really mean? Does it actually refer to the 200 μl that I put earlier into the well or the 1 ml supernatant? And which one needs to be put into the indirect method formula?

EE% = (Amount of initial drug - amount free drug or supernatant) / Amount of initial drug