The overall goal of this procedure is to reproducibly generate mycobacterium tuberculosis infected macrophage aggregate structures in 96 well plate format for drug susceptibility testing using a viability dye. This method can improve the early drug development process for anti tuberculosis compounds, which is hindered by the unproductive translation of candidate compounds into the clinical setting. The main advantage of this simple, inexpensive, BSL two compatible infection model is that it recapitulates physiologically relevant cellular penetration barriers, reminiscent of tuberculosis granulomas.

This produces drug susceptibility results more predictive of in vivo efficacy. Begin this procedure by preparing green fluorescent protein expressing M.tuberculosis MC squared 6206, hereafter referred to as MTBGFP, for infection. Keep in mind that this is avirulent strain, so all work in the protocol described here can be performed in a biosafety level two facility.

For each 96 well plate to be set up centrifuge at 2.8 times 10 to the eighth MTBGFP at 3, 200 times G for five minutes in a swinging bucket centrifuge. After the spin, aspirate the supernatant and add five milliliters of RPMIC to wash the bacteria. Pipette up and down to resuspend the cell.

Then centrifuge again. After the second spin pour off the supernatant and resuspend the bacterial cells in seven milliliters of RPMIC, then vortex for 10 seconds. Next, for each 96 well plate to be set up, centrifuge seven times 10 to the sixth THP1 human monocytic cells at 250 times G for five minutes.

After the centrifugation pour off the supernatant and resuspend the THP1 cells in seven milliliters of RPMIC. Next prepare a 96 well plate for infection by adding 200 microliters of sterile water to the wells in rows A and H and columns one and 12. This water rim will prevent the evaporation of culture medium.

Add 200 microliters of RPMIC to column two, B two to G two, for the background control or blank. To infect the THP1 monocyte use a pipette to transfer the entire prepared MTBGFP bacterial suspension to the THP1 cell suspension and mix will by pipetting up and down. The final THP1 cell density is five times 10 to the fifth per milliliter and the corresponding well duplicity of infection is 40.

Next, pour the THP1 MTBGFP suspension into a 25 milliliter reservoir, and then using a multichannel pipette, add 200 microliters of THP1 MTBGFP suspension all of the remaining 96 wells, E three through G 11. Incubate the plate at 37 degrees Celsius, with 5%CO2 for seven to 10 days. Every two days throughout the incubation, use a multichannel pipette to change the medium by slowly removing 100 microliters of spent medium from the top of each well and gently adding 100 microliters of pre warmed RPMIC.

When changing the medium it is important to take care not to disturb the MTB macrophage aggregates on the bottom of the wells. Each day visually examine the wells using a fluorescence microscope, equipped with bright field and GFP filter sets with a four to 10 X objective. Note the size of MTB macrophage aggregates and capture images if desired.

By day seven to 10 MTB macrophage aggregates should be sufficiently large to proceed for drug efficacy testing, as described later in the video. For drug testing, prepare two anti tuberculosis drugs in triplicate on a new 96 well plate as follows. First, add 125 microliters of 7H9C medium to wells B two through G 10.

Next prepare the two drugs at double the highest desired final concentration in one milliliter of 7H9C. Using a pipette add 250 microliters of each drug to wells B, C, and D 11 and then E, F, and G 11 respectively for triplicate treatments. Next, using a multichannel pipette serially dilute the test drugs two fold by moving 125 microliters from B 11 through G 11 into B 10 through G 10.

Mix by pipetting five times at each step. Continue to move 125 microliters from column to column right to left across the plate, and stop after column four. After mixing column four discard 125 microliters into a waste container.

Columns two and three should not contain any drugs. This will allow them to be used as a background, and for positive growth controls. Next retrieve the 96 well plate containing the MTB infected macrophages from the incubator.

Without tilting the plate, use a multichannel pipette to remove 150 microliters of medium from wells B two through G 11. Then tilt the plate as shown here, and insert the pipette tips into the bottom edge of the wells and remove the remaining medium, about 50 microliters. As MTB macrophage aggregates adhere to the bottom of the well, no material should be lost.

However, removal of the medium should be as gentle as possible and care must be taken to avoid resuspension during this process. Gently add 100 microliters of 7H9C medium to wells B two through G 11 of the plate, which contain the MTB macrophage aggregates. Using a multichannel pipette transfer 100 microliters of the drug containing 96 well plate to the corresponding wells of the infection plate.

Then place the plate in a sealed bag and incubate it for three days at 37 degrees Celsius. The resazurin assay relies on the oxidative species produced by metabolically active MTB to convert the blue resazurin to fluorescent pink resorufin. The change in color and fluorescence can be used as a surrogate marker to determine the amount of bacterial growth.

Prepare a stock solution of resazurin at a final concentration of 8 milligrams per milliliter in water. Filter through a 22 micrometer pore size PVDF membrane for sterilization. Prepare a working solution in resazurin by mixing the stock solution, water, and Tween-80 in a two to one to one ratio.

Final concentrations are 4 milligrams per milliliter resazurin, and 5%Tween-80. Using a plate reader, set up a program to read the fluorescence at 530 nanometer excitation and 590 nanometer emission for 24 hours every 30 minutes at 37 degrees Celsius. Pre warm the plate reader to 37 degrees Celsius.

Using a multichannel pipette, add 20 microliters of resazurin working solution to wells B two through G 11 of the drug treated plate. Place the plate on the reader and start the program. To confirm the robustness of adapting this infection model to 96 well plate format the susceptibility of MTB rifampicin RIF in moxifloxacin moxy was assessed as described in this video.

As shown here, MTB macrophage aggregate structures were successfully generated in a 96 well plate format thereby enabling through put compatibility. To quantitatively measure the conversion of resazurin as an indicator of bacterial growth fluorescence of individual wells was monitored kinetically for 24 hours. The presence of viable MTB cells is determined by the conversion of the blue resazurin dye to its pink reduced form, which is quantitatively reflected by the relative fluorescence z nets at the time point of saturation.

These representative mini graphs show the resulting fluorescence units shown on the Y axis versus time shown on the X axis. To generate drug susceptibility killing curves rifampicin and moxifloxacin treated wells were normalized to the no drug control maximal MTB growth as 100%survival. Background control blank signals were subtracted from every sample well.

The percent survival was plotted for each individual concentration of drug treatment to generate a killing curve. These data show that the minimal inhibitory concentration defines the lowest concentration of the antibiotic at which 90%growth inhibition is observed is greater than two micrograms per milliliter, for both rifampicin and moxifloxacin against MTB derived from our infection model. As such, the minimal inhibitory concentration of these two drugs against MTB using our infection model and assay is more reflective of the activity of these drugs in vivo.

After watching this video, you should have a good understanding of how to reliably and reproducibly generate MTB infected macrophage aggregate structures for drug susceptibility testing using the resazurin assay. Following this procedure modifying the drug template from two drugs as shown to a 58 drug library panel is easily performed to enable high through put drug screening.