The main advantage of this protocol is that it is less labor-intensive and time-consuming than the traditional method of counting colony forming units to estimate the intracellular growth of Mycobacterium tuberculosis. This technique makes it easier for researchers to rapidly screen FDA-approved drugs with the aim of repurposing them as host directed therapies to treat tuberculosis. The method is very simple and straightforward.
Basic knowledge of mycobacterial and EO carrier T-cell culture techniques would be enough to follow the protocol step by step. To begin, transfer six to eight milliliters of attenuated mycobacterial culture H37Ra from the T25 flask into a 15 milliliter polypropylene tube. Centrifuge the tube in a benchtop centrifuge.
After carefully removing the tube, transfer the tube to the biological safety cabinet and wait one minute for the bacteria to settle. Pour the supernatant into the disinfectant discard container. Then, recap the tube and suspend the bacteria in the remaining medium by tapping the side of the tube gently and allowing the bacteria to settle for one minute.
Add and mix two milliliters of pre-warmed complete RPMI and transfer to a 50 milliliter conical tube. To resuspend the mycobacteria, draw up the suspension in a five milliliter syringe equipped with a 25 gauge needle. Then, eject very gently down the side wall of the tube to minimize the aerosol production, and repeat the process six to eight times.
Dispose of the needle and syringe in a sharps container in the biosafety cabinet. Transfer the suspension to a two milliliter microcentrifuge tube and centrifuge to pellet down any remaining clumps. Return the tube to the safety cabinet and allow the bacteria to settle for one minute.
Transfer the upper 1 to 1.5 milliliters of the supernatant to a new tube. Discard the original tube in the waste bucket containing the disinfectant. Mix well and add various amounts of the mycobacterial suspension to the two well glass chamber slides, and incubate the slides for three hours at 37 degrees Celsius.
After pipetting up and down three times to dislodge non-phagocytosed bacteria, remove the medium from the glass chamber slide. Wash once with two milliliters of PBS. Thaw the aliquots of 4%PFA in PBS stored at minus 20 degrees Celsius.
Dilute the aliquot to 2%PFA and add two milliliters to each well. After incubating for 10 minutes at room temperature, remove the slide from the safety cabinet for staining. Discard PFA in the PFA chemical waste container and wash the slide under a gentle stream of tap water.
Using a plastic transfer pipette, dispense enough auramine to cover the cells on the slide, and incubate the slide for one minute at room temperature in the dark. Wash excess dye off the slide with tap water and add the quencher for one minute in the dark. After washing off the excess quencher, incubate for 15 minutes at room temperature with Hoechst stain in the dark.
Then wash off Hoechst stain. And after draining excess water, add a drop of antifade and a cover slip. Examine the air-dried slide under the fluorescent microscope using the 100x oil objective.
Mycobacteria will fluoresce green under Fitz filter and nuclei will fluoresce blue under DAPI filter. Count the number of mycobacteria phagocytosed per cell and the percentage of infected cells to determine MOI. Calculate the volume of mycobacterial suspension needed to achieve the required MOI based on the surface area of a well in the plate.
After mixing the mycobacteria suspension, add the calculated volume to the cells on 12 well plates to achieve the desired MOI. Incubate the plate at 37 degrees Celsius for three hours to allow mycobacteria to be phagocytosed. Then remove the extracellular bacteria by washing the wells with warm RPMI several times.
Add 500 microliters of lysis buffer for 10 minutes to lyse the macrophages in one well of the three-hour sample, and collect the lysate to determine the baseline infection rate. Then add fresh complete RPMI and required drug doses, or vehicle control, to the remaining wells. Incubate the plates in the carbon dioxide incubator at 37 degrees Celsius for one to eight days, depending on the experiment design.
To determine the percentage time to positivity, or TTP, of the initial inoculum of the three-hour sample, warm Middlebrook broth and instrument culture bottles to room temperature. Transfer the medium from the 12-well plate to the corresponding labeled conical tubes and add 500 microliters of sterile lysis buffer to each well. After 10 minutes, gently scrape the cells from the well with a sterile scraper and combine them with the medium in the appropriate conical tube.
Once the well is washed with 0.5 milliliters of sterile PBS and combined with the medium and lysate, break the clumps by gently passing each sample through a 25 gauge needle syringe six to eight times. Dilute the sample in MB broth by adding 900 microliters of MB broth to 100 microliters of sample, and repeat the harvest process at the required time points during incubation. Prepare the instrument cluster bottles by sterilizing the rubber cap with 70%alcohol.
Transfer enough nutrient supplements for all the samples into a conical tube, and use a needle and syringe to inject 0.5 milliliters of nutrient supplement into the instrument culture bottle. Then, pipette 500 microliters of the one-in-10 diluted sample in a one milliliter v-bottom tube. Use a needle and syringe to inject the 500 microliters of sample into the assigned instrument culture bottles.
After carefully transporting the bottles from the biosafety cabinet to the instrument for loading, press the loading"button on the automated microbial detection system. Scan the barcodes on instrument culture bottles and place the bottles into the detection system incubator at 37 degrees Celsius for up to 42 days. Read and record the time taken to reach positivity from the instrument screen and calculate percentage TTP.
A positive TTP indicates mycobacterial growth. The automated liquid culture instrument monitored carbon dioxide levels every 10 minutes and calculated TTP, which is the number of days from inoculation until cultures are flagged as positive. An inverse relationship between TTP and log(10)values of CFU in the initial inoculum was illustrated.
When intracellular growth of Mycobacterium tuberculosis within macrophages determined by enumerating CFU was compared to the automated culture method described, a significant correlation between results was obtained. Mycobacterium tuberculosis growth was graphically presented by comparing the TTB values for up to eight days after infection of macrophages to the initial TTP value. A similar trend between CFU and liquid culture demonstrated significant inhibition of Mycobacterium tuberculosis growth in macrophages in the presence of all-trans retinoic acid, or AtRA, in solution, or the equivalent dose of AtRA encapsulated in PLGA microparticles.
A dose-response experiment was carried out in infected THP-1 cells to determine the efficacy of the antibiotic Linezolid alone, or the combination of linezolid and interferon gamma. No significant interaction was observed between the drugs. AFP staining allows us to use a low MOI to minimize cell death when infecting macrophages, and to ensure that the same MOI is used regardless of the treatment used.
Following this protocol, lead anti-TB drugs can be identified and studied further to investigate their in vivo efficacy.
