In cell analyzer 6500 hs

Localization of Ncr1-Cub-TF was investigated via Yellow Fluorescent Protein (YFP) fusion as previously described (Snider et al. 2010 (link)). The Ncr1-Cub-YFP-TF strain, constructed using the L3 plasmid, was grown in selection broth (YPD + G418) overnight at 30°C with agitation, diluted to a starting cell density of OD₆₆₀ = 0.2 in 3 mL of YPD, incubated at 30°C with agitation for 5 h, centrifuged, washed with ddH₂O, and resuspended in 30 µL ddH₂O. For imaging, 1 µL of cells was mounted onto a microscope slide and imaged using an IN Cell Analyzer 6500 HS (General Electric) confocal microscope with a 60 × objective lens. Differential interference contrast (DIC) was used to capture cell shape and vacuole; YFP was detected using the green filter excited at 488 nm and emitted at 524 nm with 1 s exposure. Images were prepared using FIJI ImageJ.

Human PBMC were thawed one day prior to assay. The day after, an extracellular matrix (ECM) was prepared by mixing 150 µL of 10× PBS (D1408, Sigma-Aldrich, St. Louis, MO, USA), 900 µL of type I collagen solution (3 mg/mL) (5005, Advanced BioMatrix, San Diego, CA, USA), 6 µL of 0.1% fibronectin (F0985, Sigma Aldrich, St. Louis, MO, USA) and 423 µL of Milli-Q water per 1.5 mL of matrix solution and kept on ice until use. Immediately before use, 21 µL of 1 M NaOH (pH 7.4) was added and mixed slowly, and the final pH of the solution should be ~7.0. Afterwards, PBMC were mixed at room temperature with ECM mixture at 2.5 × 106 cells/250 µL well of a 96-well plate. Ten µg/mL of recombinant Mtb CFP10: ESAT6 chimera protein (DAGA-193, Creative Diagnostics, Shirley, New York, NY, USA) was added to the ECM for sample infection with 25 mM HEPES, 2 mM of L-glutamine and 20% of heat-inactivated type AB human serum at 37 °C. The formation of granulomas was evaluated after 2 and 5 days of culture and captured using IN Cell Analyzer 6500 HS (GE Healthcare Life Science, Piscataway, NJ, USA). Only multilayered structures containing ~4–8 cell layers were considered granulomas. On day 5, granulomas were collected with manual pipette and incubated with 0.05% Trypsine-0.53 mM EDTA for 15 min at 37 °C to dissociate cells. Full culture details are provided in the online Supplementary Materials.

The Ncr1-mScarlet-I Cyb5-GFP strain was grown overnight at 30°C with agitation, diluted to a starting cell density of 0.255 × 10⁷ cells/mL in 2 mL of SC, incubated at 30°C with agitation for 5 h, centrifuged, washed with PBS, and resuspended in 50 µL PBS. For imaging, 1 µL of cells was mounted onto a microscope slide and imaged using an IN Cell Analyzer 6500 HS (General Electric) confocal microscope with a 60 × objective lens. DIC was used to capture cell shape and vacuole, GFP was detected using the green filter excited at 488 nm and emitted at 524 nm with a 2 s exposure, and the mScarlet-I was detected using the orange filter excited at 561 nm and emitted at 605 nm with a 3 s exposure. Images were prepared using FIJI ImageJ.

The cellular supernatants were removed after 12 h of PAM culture, and the cells were washed with PBS. Each well received 50 μL of PBS that contained 10 μg/mL propidium iodide and 10 μg/mL Hoechst 33342 for a 30‐min incubation period at 37°C. Incubated cells were then scanned using an In‐Cell Analyzer 6500HS (GE Healthcare, USA, 10× objective). Propidium iodide (PI) and Hoechst 33342 staining were identified at a wavelength of 642 nm and 405 nm, respectively. Live/Dead cell viability analysis was performed using IN Carta Image Analysis Software.¹⁰ Cell viability is calculated as follows: cell count (PI)/cell count (Hoechst 33342) × 100%.