Generation of bone marrow- derived macrophages Bone marrow- derived macrophages (BMDM) were obtained as previously described  with some modifications. TSA HDAC Briefly, bone marrow cells were flushed from the femur of eight- to ten- week-old specific pathogen-free C57BL/6 mice. The cells were dispersed in Dulbecco’s
modified Eagle’s medium, DMEM (Sigma, St Louis, MO), supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 M 2-mercaptoethanol (Gibco BRL, Grand Island, NY), 10% heat-inactivated FBS (Hyclone, Road Logan, UT), 50 μg/ml gentamicin (Gibco BRL, Grand Island, NY), and cultured at 37°C in 5% CO2 atmosphere. Nonadherent cells were collected after 18 h, resuspended in the complete DMEM, supplemented with 20% L929 cell-conditioned medium as a source of M-CSF, and cultured for 7 days, replacing the medium
on day 3. The monolayer cells were scraped, resuspended in DMEM, supplemented with 2% FBS, without antibiotics, and plated at a concentration of 5 x 105 cell/ml in a 96-well plates, 100 μl/well. Treatment with cytokines and infection of cell cultures The BMDM cultures were incubated overnight, pretreated, or not, with murine recombinant IFN-γ, 100 U/ml (Bioscience, Camarillo, CA), or IL-10, 20 ηg/ml (Bioscience, Camarillo, CA) for 2 h, and infected with single-cell suspensions of mycobacterial strains at MOI 1:1 and 5:1. learn more After 3 h of incubation at 37°C, infected monolayers were washed and incubated for click here additional 6 d in new aliquots of culture medium. In the pretreated cultures, the cytokines were renewed and were present throughout the incubation period. Cell viability of infected MΦ was monitored
by trypan blue exclusion and was over 90% in all experiments. Quantification of mycobacterial growth in macrophages Mycobacterial ability to grow intracellularly was evaluated by colony-forming units (CFU) test in the MΦ cultures infected at a MOI of 1:1. After 0, 3 and 6 d of incubation, cells were lysed with 1% saponin isometheptene to release intracellular bacteria. Lysates of infected cells were resuspended, transferred into screw caps, vortexed and sonicated in a preheated waterbath sonicator (Unique 800, Brazil) at 37°C for 2 min. Aliquots of the sonicate were diluted 10-fold in PBS, plated in quadriplicates on 7 H10 agar plates and incubated at 37°C for 21 days. Cytokine quantification To study cytokines secreted by infected MΦ, the cell cultures were infected at a MOI 5:1 in the presence or absence of recombinant IFN-γ and IL-10, as indicated above. The infected monolayers were washed and incubated for additional 48 h. After incubation, the culture supernatants were collected, filtered through 0.22 μm Spin-X centrifuge tube filters (Corning, NY), and the supernatant aliquots were stocked at −70°C for posterior cytokine determination.