An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model
Published: January 31, 2024
Abstract
Source: Schaaf, K., et al. A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis. J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.
Protocol
1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP) NOTE: The M. tuberculosis H37Rv derived auxotroph strain mc26206 (ΔpanCD, ΔleuCD) transformed with the gfp (green fluorescent protein) expressing plasmid pMN437 is used throughout this protocol. It is possible to substitute the Mtb-GFP strain with non-gfp expressin…
Representative Results
Figure 1: Drug plate layout. (A) Example template of the drug challenge plate used in step 4. This allows for the parallel testing of two drugs in triplicate wells at 8 defined concentrations. As a representative experiment, we used rifampicin (RIF) and moxifloxacin (MOXI) starting at 2 µM and 2.5 µM, respectively. (B) An alternative template for throug…
Divulgaciones
The authors have nothing to disclose.
Materials
| 7H9 | BD Difco | 271310 | Follow manufacturer's recommendations |
| Middlebrook OADC | BD Biosciences | 212351 | |
| Tyloxapol | Sigma | T8761 | Prepare 20% stock solution in H2O; filter sterilize |
| D-Pantothenic acid hemicalcium salt | Sigma | P5710 | Prepare 24 mg/ml stock solution in H2O; filter sterilize |
| L-leucine | MP Biomedicals | 194694 | Prepare 50 mg/ml stock solution in H2O; filter sterilize |
| Hygromycin B | EMD Millipore | 400051 | Prepare 200 mg/ml stock solution in H2O |
| Nalgene Square PETG media bottle | Thermo Fisher | 2019-0030 | |
| RPMI 1640 media | Hyclone | SH30027.01 | |
| Fetal Bovine Serum | Atlanta Biologicals | S12450H | |
| L-glutamine | Corning | MT25005CI | |
| HEPES | Hyclone | SH30237.01 | |
| Cytation 3 plate reader | Biotek | Interchangable with any fluorescent plate reader and microscope | |
| Gen5 Software | Biotek | Recording and analysis of rezasurin coversion | |
| Rifampicin | Fisher Scientific | BP2679250 | Prepare 10 mg/ml stock solution in H2O |
| Moxifloxacin Hydrochloride | Acros Organics | 457960010 | Prepare 10 mg/ml stock solution in H2O |
| Resazurin Sodium Salt | Sigma | R7017 | Prepare 800 μg/mL stock solution in H2O; filter sterilize |
| Tween-80 | Fisher Scientific | T164500 | Prepare 20% stock solution in H2O; filter sterilize |
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Citar este artículo
An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model. J. Vis. Exp. (Pending Publication), e21926, doi: (2024).