Rmm csf

M. tuberculosis strain H37Rv-GFP⁴² was cultured at 37 °C in Middlebrook 7H9 broth supplemented with 10% Middlebrook OADC, 0.2% glycerol, and 0.05% Tween-80. Murine bone marrow-derived macrophages (BMDMs) were prepared as previously described^{43 (link)} from C57BL6 mice (Jackson Laboratories) in accordance with protocol number 2007N000048 approved by Massachusetts General Hospital Institutional Animal Care and Use Committee. For infections, BMDMs were seeded in 6 well plates overnight in DMEM supplemented with 25 ng/ml rmM-CSF (R&D Systems). M. tuberculosis was grown to mid-log phase, washed once in PBS and resuspended thoroughly. A low-speed spin (500 rpm) was performed to pellet clumps. Bacteria were added to DMEM with 20% heat-inactivated horse serum to yield a multiplicity of infection of 1. After 4 hours, extracellular bacteria were removed with washes and fresh media was added. All M. tuberculosis infections were conducted using BL3 practices and containment equipment according to protocol IBC-2016-00095-1 approved by the Institutional Biosafety Committee of the Broad Institute.

Precision-cut liver slices (PCLS) were prepared from the whole liver with a Krumdieck tissue slicer (Alabama Research and Development). The PCLS had the following characteristics: diameter, 5 mm; thickness, 250–300 um; weight, 4 to 5 mg. The PCLS were incubated individually in 12-well plates in 1.3 mL of Williams Medium E (Gibco, #12551-032) supplemented with 10% Special HI fetal calf serum (FCS; Gibco, #16140-071), 25 mM D+glucose (Sigma, #68769-100ML), 2 mM GlutaMAX™ (Gibco, #31966-021), and 50 µg/mL gentamycin (Invitrogen). The PCLS were exposed to 100 ng/mL rmM-CSF (R&D Systems, #416-ML/CF), 118 ng/mL rmIL-4 (R&D Systems, #404-ML-010/CF), 256 ng/mL rmIL-13 (R&D Systems, #413-ML-005/CF), 20 ng/mL rmTNFα (R&D Systems, #410-MT/CF), and 100 ng/mL PMA (Sigma, #P8139). The PCLS were cultured for the indicated times in an incubator (Binder, Tuttlingen, Germany) at 37°C, with 90% O₂ and 5% CO₂, and horizontally shaken at 60 rpm.
The viability of PCLS was assessed by quantification of adenosine triphosphate (ATP) using a bioluminescence kit (Roche #11699695001). The obtained ATP value (pmol) was normalized to the total protein content (μg, measured with the Lowry method).

Murine bone marrow-derived macrophages (BMDM) were prepared as previously described [17 (link)] from C57BL6 mice (Jackson Laboratories). BMDM were seeded in 24 well plates overnight in DMEM with 20% FBS and 25ng/ml rmM-CSF (R and D Systems). Mtb strains were grown to mid-log phase, then used to infect BMDM at an MOI of 2:1. After 4 hours of phagocytosis, cells were washed once with PBS and media was added back. RNA was harvested 24 hours after infection with TRIzol (ThermoFisher Scientific) and prepared according to the manufacturer’s protocol. cDNA was prepared using SuperScript III (ThermoFisher Scientific) according to the manufacturer’s protocol. qPCR was performed using primers specific to the indicated genes.

Total bone marrow cells were isolated from the femurs of 12-week-old mice and were seeded in Petri dishes at a density of 10⁶ cells/mL in 10 mL of α-minimum essential medium (αMEM) (Thermo Fisher Scientific), supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin in the presence of 30 ng/mL recombinant mouse (rm)M-CSF (R&D Systems). At day 3 of culture, cells were trypsinized and seeded in 96-well plates at a density of 25 × 10⁴ cells/well in αMEM supplemented with 30 ng/mL of rmM-CSF and 20 ng/mL rmRANKL (R&D Systems). The culture medium was refreshed after 3 days. After 4 days of differentiation in the presence of RANKL, cells were fixed with 4% PFA and stained for tartrate-resistant acid phosphatase (TRAP), using the Leukocyte Acid Phosphatase Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Cells with three or more nuclei were considered osteoclasts and were further scored as small osteoclasts with 3–5 nuclei and bigger osteoclasts with ≥6 nuclei.