An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

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 expressing wild-type strain to enable easier accessibility of the strain for researchers. However, gfp expression is desirable to enable visual confirmation of phagocytosis and the formation of Mtb/macrophage aggregates. For long-term storage, Mtb-GFP were frozen at -80 °C in complete 7H9 media (described in step 1.1) supplemented with 20% glycerol.

1. Prepare complete Mtb-GFP media (7H9-C) by supplementing 7H9 medium with 10% Middlebrook OADC (Oleic Albumin Dextrose Catalase), 0.02% tyloxapol, 24 µg/mL D-pantothenic acid, 50 µg/mL L-leucine and 50 µg/mL Hygromycin B.
2. Thaw one vial of Mtb-GFP and add 10 mL of 7H9-C in a 30 mL square bottom PETG flask.
3. Incubate at 37 °C on a rotary shaker (50 rpm). Take optical density measurements (OD₆₀₀) every few days until OD₆₀₀ reaches 1.0 (approximately 5-8 days).
4. Dilute and passage culture as needed to maintain an OD₆₀₀ between 0.2-1.0.
5. For infection, use an established culture of Mtb-GFP within the logarithmic growth phase (OD₆₀₀ of 0.6-1). To avoid clumpy cultures, sonicate the Mtb culture flask in a water bath (130 W; 3 x 5 s pulses) once or twice a week.

2. Culture Conditions for THP-1 Cells

1. Prepare complete THP-1 cell media (RPMI-C) by supplementing RPMI 1640 medium with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
2. Incubate cells in a T-75 flask at 37 °C in a humidified atmosphere of 5% CO₂.
3. Count cells using a hemocytometer or flow cytometry every other day and maintain THP-1 cells at a density between 0.1 to 0.6 million per mL.

3. Infection Protocol to Generate Mtb/Macrophage Aggregate Structures

1. THP-1 cell preparation (per 96-well plate)​
   1. Centrifuge 7 x 10⁶ THP-1 cells at 250 x g for 5 min. Resuspend in 7 mL RPMI-C.
2. Mtb-GFP preparation​
   1. Centrifuge 2.8 x 10⁸ Mtb-GFP at 3,200 x g for 5 min in a swinging-bucket centrifuge using a conversion of OD₆₀₀ 1.0 = 3 x 10⁸ bacteria/mL.
   2. Wash once with RPMI-C and centrifuge as in step 3.2.1.
   3. Resuspend in 7 mL RPMI-C and vortex for 10 s.
3. Infection
   ​NOTE: See Figure 1 for the template.
   1. Prepare a 96-well plate by adding 200 µL of sterile water in rows A and H and columns 1 and 12 for a water rim to prevent evaporation of the culture medium.
   2. Add 200 µL RPMI-C to column 2 (B2 to G2) for the background control (Blank).
   3. To infect, add Mtb-GFP suspension (step 3.2) to THP-1 cell suspension (step 3.1) and mix well by pipetting. The final THP-1 cell density is 5 x 10⁵ per mL and the corresponding multiplicity of infection is 40.
   4. Pour the THP-1/Mtb-GFP suspension into a 25 mL reservoir.
   5. Add 200 µL of THP-1/Mtb-GFP suspension to all remaining 96 wells (B3 through G11) using a multi-channel pipette.
      NOTE: If working with multiple 96-well plates, regularly resuspend the remaining THP-1/Mtb suspension in the reservoir to ensure an even mixture is added to each well.
   6. Incubate at 37 °C with 5% CO₂ for 7-10 days.
   7. Change media every 2 days by slowly removing 100 µL spent media from the top of each well and gently adding 100 µL pre-warmed RPMI-C using a multi-channel pipet. Do not resuspend wells and disturb the Mtb/macrophage aggregates on the bottom of the wells.
   8. Visually examine the wells by fluorescence microscopy (4-10X objective) daily, taking note of the size of Mtb/macrophage aggregates. By day 7-10, Mtb/macrophage aggregates should be sufficiently large (refer to Figure 2 for reference) to proceed with drug efficacy testing (Section 4).
   9. If desired, capture images of the 96 wells using an automated cell imaging system fitted with bright field and GFP filter sets to document Mtb/macrophage aggregate formation.
      NOTE: If users do not have access to an automated imaging system, images of representative wells can be taken using any appropriate microscope with GFP and bright field capabilities.

4. Growth Inhibition Assay to Assess Drug Efficacy Against Mtb Derived from Mtb/Macrophage Aggregates

1. Drug preparation (triplicate conditions for two drugs)
   NOTE: See Figure 1A for the template.
   1. In a separate 96-well plate, add 125 µL of 7H9-C media to B2 through to G10.
   2. Prepare two drugs at double the highest desired final concentration in 1 mL of 7H9-C to account for the dilution in step 4.2.4.
   3. Add 250 µL of each drug to wells B11-C11-D11 and E11-F11-G11, respectively for triplicate treatments.
   4. Using a multi-channel pipet, serially dilute the test drugs two-fold by moving 125 µL from B11-G11 into B10-G10. Mix by pipetting 5 times at each step.
   5. Continue to move 125 µL from column to column (right to left) across the plate, and stop after column 4.
   6. After mixing column 4, discard 125 µL into a waste container. Columns 2 and 3 should not contain any drugs to allow for use as a background (Blank) and positive growth controls.
      NOTE: Alternatively, follow the template in Figure 1B for testing drug libraries. Each 96-well plate can accommodate 58 drugs at a single concentration. Prepare this drug plate using double the desired final concentration since it will be diluted in half in step 4.2.4.
2. Drug treatment:
   1. Retrieve the 96-well plate containing the Mtb-infected macrophages (Mtb/macrophage aggregates).
   2. Carefully decant all the relevant wells (B2 through G11) with a multi-channel pipet. Perform this in a two-step manner: first, remove 150 µL without tilting the plate, and then remove the remaining media (~50 µL) by tilting the plate and inserting the pipette tip into the bottom edge of the well. As Mtb/macrophage aggregates are adherent to the bottom of the well, no material should be lost.
   3. Gently add 100 µL of 7H9-C media to all relevant wells (B2 through G11) of the plate containing the Mtb/macrophage aggregates.
   4. Using a multi-channel pipet, transfer 100 µL from the drug-containing 96-well plate (step 4.1) to the corresponding wells of the infection plate.
   5. Place in a sealed bag and incubate for 3 days at 37 °C.

5. Quantification of Drug Efficacy Using the Resazurin Microtiter Assay

1. Prepare resazurin stock solution at a final concentration of 0.8 mg/mL in H₂O. Filter through 0.22 µm pore size polyvinylidene fluoride (PVDF) membrane for sterilization.
2. Prepare resazurin working solution by mixing resazurin stock solution, H₂O, and Tween-80 (20% solution in H₂O) in a 2:1:1 ratio. The final concentrations are 0.4 mg/mL resazurin and 5% Tween-80.
3. Using a plate reader, set up a program to read fluorescence at 530 nm excitation and 590 nm emission every 30 min for 24 h at 37 °C. Pre-warm plate reader to 37 °C.
4. Add 20 µL of resazurin working solution to all relevant wells (B2 through G11) of the drug-treated plate (step 4.11) using a multi-channel pipet.
5. Place the plate on the plate reader and start the program set up in step 5.3.
   NOTE: To facilitate the processing of high volumes of assay plates, it is sufficient to develop the assay in a 37 °C incubator and perform single fluorescence reads every 24 h. This is also applicable to users who do not have access to a plate reader with kinetic and incubation capabilities.