Syto 24

For flow cytometry analysis, 10-fold serial dilutions of luminal and mucosal samples were prepared in anaerobic Dulbecco’s Phosphate-buffered Saline (DPBS) (Sigma-Aldrich, Bornem, Belgium) and stained with 0.01 mM SYTO24 (Life Technologies Europe, Merelbeke, Belgium) for 15′ at 37 °C in the dark. Samples were analyzed on a BD Facsverse (BDBiosciences, Erembodegem, Belgium) using the high-flow-rate setting. Bacteria were separated from medium debris and signal noise by applying a threshold level of 200 on the SYTO channel. Flow cytometry data were analyzed using FlowJo, version 10.5.2.

All staining was performed in 96-well plates with 50 µL of fixed Mtb cells diluted in 50 µL of PBST. Staining was performed with 0.6 μg of FM4-64FX (ThermoFisher; F34653) and 15 μL of a 0.1 μM SYTO 24 (ThermoFisher; S7559) stock in each well containing PBST and fixed bacilli. The plate was then incubated at room temperature in the dark for 30 min. Once stained, the cells were washed once with an equal volume of PBST and resuspended in 30 µL of PBST. Stained Mtb were spotted onto agar pads (1% wt/vol agarose; SigmaAldrich; A3643-25G). Images were captured with a widefield DeltaVision PersonalDV (Applied Precisions) microscope. Bacteria were illuminated using an InsightSSI Solid State Illumination system with transmitted light for phase contrast microscopy. SYTO 24 was imaged using Ex. 475 nm and Em. 525 nm. FM4-64FX was imaged with Ex. 475 nm and Em. 679 nm. Montage images were generated using a custom macro that captures 25 individual fields of view per image. Two technical replicate images were taken from each sample for a total of 50 images per biological replicate. Three biological replicates were generated for each drug treatment. Images were recorded with a DV Elite CMOS camera for all three channels.

The Seahorse XF Real-Time ATP Rate Assay (Agilent, 103592) was performed using the Seahorse XFe96 Analyzer. Cells were seeded at 5000 cells per well in an XF cell plate coated with 0.05% poly-L-lysine and then incubated overnight. Every 24 h for 72 h total, cells were incubated with fresh DMSO or 40 µM DGAT1 inhibitor in nutrient-limited media (DMEM supplemented with 5 mM glucose, 1 mM glutamine, 1 mM sodium pyruvate, and 1% FBS). Cells were incubated in assay medium (Seahorse XF DMEM medium, pH 7.4, 10 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate) for 1 h prior to measurement in a CO₂-free incubator at 37 °C. During assay run, cells were exposed to 1.5 µM oligomycin and 0.5 µM rotenone/antimycin A. OCR, ECAR, and Proton Efflux Rate were normalized to nuclei FU via SYTO 24 (Thermo Fisher, S7559). Experimental measurements were analyzed using the Agilent Wave software generating the mitoATP and glycoATP measurements.

Mtb Ara-LAM was obtained from BEI Resources and used at 5 µg/mL. Mtb H37Rv genomic DNA was obtained from 28 days colonies growing in Löwenstein–Jensen medium by CTAB method as previously described (Yamashiro et al., 2016 (link)). Recombinant human (rh) IL-6 was purchased from Immunotools. Anti-IFNAR2A (clone MMHAR-2, PBL) and anti-IFN-γ (clone B27, Immunotools) neutralizing antibodies were used at 1 and 10 µg/mL, respectively and anti-IL-6R (Tocilizumab, Roche) was used at 1 µg/mL. Fluorescent dye Syto24 was obtained from Thermo Fisher Scientific.

Fresh AM, and VLAM and DLAM post-rehydration were stained with SYTO 24 and Ethidium Homodimer-1 (Thermo Fisher Scientific, Waltham, MA). A working LIVE/DEAD^® nuclear stain was prepared by adding 1 μL of reconstituted SYTO 24 solution and 3 μL of reconstituted Ethidium Homodimer-1 solution per 1 mL DPBS. Sixteen-millimeter-diameter disks were cut from 25 cm² samples of fresh AM, VLAM, and DLAM and they were used for staining. Samples were completely submerged in the staining solution for 5 min. They were rinsed in DPBS to remove excess staining, and cell viability was evaluated by using Invitrogen™ EVOS™ FL Auto Imaging System System and Celleste™ Image Analysis Software (Thermo Fisher Scientific).

The Seahorse XF Mito Stress Test (Agilent, 103015) was performed using the Seahorse XFe96 Analyzer. For melanoblasts and melanocytes, cells 30,000 melanoblasts and 10,000 melanocytes were seeded per well in an XF cell plate as previously described^{13 (link)}. For human A375 cells, cells were treated with DMSO, IBMX, or Forskolin as described above then cells were trypsinized and resuspended in drug-containing media at 30,000 cells per well in an XF cell plate coated with 0.05% poly-L-lysine then incubated overnight (Sigma-Aldrich, 4707).
Cells were incubated in XF Mito Stress Test assay medium (Seahorse XF DMEM medium, pH 7.4, 10 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate) for 1 h prior to measurement in a CO₂ free incubator at 37 °C. During assay run, cells were exposed to 2.0 µM oligomycin, 2.0 µM FCCP, and 0.5 µM rotenone/antimycin A. OCR and ECAR were normalized to nuclei fluorescence unit (FU) via SYTO 24 (Thermo Fisher, S7559) or protein via Pierce BCA Protein Assay Kit (Thermo Fisher, 23227) as indicated. Experimental measurements were analyzed using the Agilent Wave software.