Measuring Drug Binding to Caseum from Tuberculosis Lesions via the Rapid Equilibrium Dialysis Method

Take a homogeneous suspension of caseum-the necrotic, lipid-rich material from tuberculosis lesions, which harbors drug-tolerant mycobacteria at its core.

Add the test drug to the homogenate and vortex to facilitate its interaction with the caseum matrix.

Place a rapid equilibrium dialysis insert-containing donor and receiver chambers separated by a semi-permeable membrane-into the base plate.

Load the drug-spiked homogenate into the donor chamber and add buffer to the receiver chamber.

Seal the plate and incubate with rotation.

During incubation, the free drug diffuses across the membrane, reaching equilibrium between both chambers.

Once equilibrium is reached, dilute the donor sample with buffer and the receiver sample with drug-free homogenate to normalize matrix conditions for drug quantification.

Finally, quantify the drug concentration and calculate the unbound drug fraction or fu.

The fu reflects the free drug available to diffuse into caseum and reach drug-tolerant mycobacteria.

Add 6.5 microliters of the prepared test compound to 643.5 microliters of the homogenate. To achieve the final concentration of five micromolar and vortex.

Next, place the red inserts into the base plate. Pre