Tb fluorescent stain kit m

Mtb retrieved from both granuloma models were pelleted by centrifugation at 6000 × g for 5 min prior to being inactivated with 1X CellFIX (BD Biosciences) for 20 min at room temperature. Fixed samples were spotted on glass slides, air dried and heat fixed at 70°C for at least 2 h. Acid-fast staining using TB Fluorescent Stain Kit M (BD) was performed in combination with neutral lipid staining dye Nile red (Sigma-Aldrich). Each sample was stained with auramine-O for 20 min, decolorized for 30 s, covered with Nile red (10 μg/ml in ethanol) for 15 min and counterstained with potassium permanganate for 2 min. Samples were gently washed with distilled water between each step. Stained slides were examined using a Leica DM5000 B fluorescence microscope. For quantitative analysis, at least 200 bacteria per sample were counted.

The kasB mutants were grown to log phase and 10 µl of culture were spread onto a glass slide. The slides were heated at 100°C for 2 min, dipped into 10% formalin for 30 min, dried and stained using the TB Fluorescent Stain Kit M (BD, Auramine staining) or the TB Stain Kit K (BD, Carbolfuchsin staining).

At the specified time-points, supernatant was removed and wells were treated with 250 μl of collagenase (1 mg/ml; Sigma) for 40 min at 37°C (5% CO₂) to release the PBMCs from the ECM. Host cells were pelleted at 400×g for 5 min and lysed with 0.1% Triton X-100 (Sigma) for 20 min at RT, followed by centrifugation at 6000×g for 5 min to obtain the Mtb pellet. Bacilli were inactivated with 1× CellFIX (BD) for 20 min at RT. Fixed samples were put on glass slides, air dried and heat fixed at 70°C. Fluorescent acid-fast staining using TB Fluorescent Stain Kit M (BD) was performed in combination with neutral-lipid staining dye Nile red (Sigma) [28 (link)]. Each sample was stained with auramine-O for 20 min, decolorized for 30 s, covered with Nile red (10 μg/ml) for 15 min and counterstained with potassium permanganate for 2 min, including gentle washes with distilled water between each step. Air-dried, stained slides were mounted using Vectashield mounting medium (Vectorlabs) and examined using a Leica DM5000 B fluorescence microscope (Leica). For quantification purposes, at least 200 bacteria per sample were counted. Representative micrographs for auramine-O and Nile red-positive Mtb are shown in S4 Fig.