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

Abstract

Source: Sarathy, J. P. et al. An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions. J. Vis. Exp. (2017)

This video demonstrates an in vitro assay for measuring drug binding to caseum from tuberculosis lesions using rapid equilibrium dialysis. It outlines the steps for preparing caseum suspensions, performing dialysis, and calculating the unbound drug fraction to predict drug penetration into caseum.

Protocol

1. Rapid Equilibration Dialysis (RED) Assay

1. Spike 6.5 µL of the 500 µM solution of the test compound into 643.5 µL of the homogenate to achieve the final concentration of 5 µM (≤1% DMSO) and vortex.
2. Place the RED inserts into the base plate. Add 200 µL of the drug-spiked matrix into the donor chamber (red ring) of each RED insert and 350 µL of phosphate-buffered saline (PBS) into each receiver chamber. Prepare 3 inserts for each test compound (triplicate samples). Seal plate with an adhesive plate seal and incubate at 37 °C on the thermomixer at 200 rpm (1 x g) for 4 h.
3. After the incubation, gently mix the contents of the donor and receiver chambers by pipetting up and down 2-3 times. Pipette out 20 µL aliquots of homogenate from the donor chambers and add to 20 µL of clean PBS in a 1.5 mL tube (1:1). Similarly, pipette out 20 µL aliquots of PBS samples from the receiver chambers and add to 20 µL of clean homogenate (matrix matching).
   NOTE: Matrix-matching eliminates the need for 2 separate calibration curves (in homogenate and PBS) to be made for quantitative analysis. Contents of the donor chamber may sediment over time. Gently mix the contents by pipetting before removing aliquots.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pierce RED Device Reusable Base Plate	Fisher Scientific	PI-89811
Pierce RED Device Inserts, 50/box	Fisher Scientific	PI-89809
Pierce RED insert removal tool	Fisher Scientific	89812
Adhesive plate seal	Fisher Scientific	08-408-240
PBS, pH 7.4, 10x 500 mL (Gibco)	Life Technologies	10010-049
DMSO	Sigma	472301