To derive macrophages from the bone marrow of wild type adult mouse, plate the bone marrow cells in a 96-well optically-treated flat bottom plate containing DMEM supplemented with 10%FBS and 10%LCCM. Incubate the cells at 37 degrees Celsius with 7%carbon dioxide for 10 days. After differentiation, pipette 75 microliters of the Mycobacterium abscessus suspension into the macrophages containing wells.
Incubate the cells at 37 degrees Celsius with 7%carbon dioxide for four hours. Using a microplate reagent dispenser, equipped with a small cassette, add 110 microliters of DMEM supplemented with 10%FBS and 10%LCCM into the compounds or solvent control columns. Dispense an additional 104 microliters of DMEM supplemented with 10%FBS and 10%LCCM into the columns containing the highest concentration of the compound or solvent.
Now pipette 5.9 microliters of the compounds or solvent into their designated wells in the columns. Using a multi-channel micropipette, perform two serial-dilution for each compound and solvent. After the last dilution, discard the excess 110 microliters of medium from the last well.
Add 110 microliters of DMEM supplemented with 10%FBS and 10%LCCM into all columns, excluding blank wells. Prepare a solution of clarithromycin at a concentration of two micrograms per milliliter in a DMEM. After incubation, remove the 96-well plate containing infected macrophages from the incubator.
Using a plate washer, wash the cells three times with 200 microliters of infection washing solution. Transfer 200 microliters of the compounds to the plate containing the infected macrophages. Then add 200 microliters of supplemented cell culture medium and clarithromycin solution to their respective wells.
Incubate the plate at 37 degrees Celsius with 7%carbon dioxide for 48 hours. After incubation, wash the infected cells three times each with 200 microliters of the compounds washing solution. Using a multi-channel micropipette, dispense 200 microliters of fixation solution into all wells and incubate for 10 minutes.
After washing the cells, transfer 200 microliters of permeable solution into all wells and incubate for 15 minutes. Next, aspirate the permeable solution before adding 100 microliters of the staining solution into all wells. Incubate for 30 minutes at room temperature.
After incubation, wash the cells three times with 200 microliters of the compound's washing solution. To screen the plate using a high-content imaging system, select the Plate Type as 96 microtiter plate. Objective as 20X with 0.4 numerical aperture and Mode to Confocal.
Afterward, select the DAPI, CellMask deep red, and mCherry channel to capture the stained nuclei, cytoplasm, and bacteria signal, respectively. Select the wells of the plate and the respective fields of each well for the analysis. Next, click on the test and capture an image to check the settings.
Finally, set up an equipment handling robot to automate image acquisition overnight. This robotic arm exchanges the plates in the high-content imaging system without human intervention. Compounds daunorubicin, doxorubicin, thiostrepton, epirubicin, and pyrvinium pamoate were toxic to the mycobacteria infected macrophages leading to less than 85%cell viability.
Compounds moxalactam and besifloxacin had less than 50%mycobacteria viability at 13.3 micromolar, but showed no significant difference from DMSO control. Compounds cefdinir, gatifloxacin, and moxifloxacin were potent against intracellular mycobacteria at 13.3 micromolar with compound pyrvinium pamoate being the most active. Compounds, roxithromycin, clarithromycin, and rifabutin showed extreme potency against intracellular mycobacteria with clarithromycin reducing mycobacterium viability to less than 10%across all concentrations.
