Macrophage-Mycobacterium Abscessus Interaction

BALB/c mice (n = 4) were euthanized and the femur bone marrow was extracted, homogenized and plated with 10 ng/mL of GM-CSF as described previously (Becker et al., 2012 (link)). After 3 days of culture, the medium was supplemented with 10 ng/mL of GM-CSF. After 7–10 days of culture, the macrophages were transferred to a 96-well plate at 10⁵ cells/well. After a 1 h incubation at 37°C in a CO₂ incubator, 10⁶ CFU/well of M. abscessus subsp. massiliense were added to the cultures [multiplicity of infection (MOI), 10:1] and further incubated for 3 h. Then, the cell cultures were washed with RPMI-1640 (HIMEDIA), containing 10% fetal bovine serum, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine, and 2 mM non-essential amino acids.
After this procedure, the infected macrophages were treated with 200 μM of NDBP-5.5 (MBC = 200 μM) prepared in RPMI-1640 media. After 72 h, the cells were treated with 200 μL of deionized water, and after homogenization, the suspensions were plated on MH agar plates for culturing to determine the number of CFU. Controls containing only infected macrophages or macrophages treated with CLR were added to all of the 96-well test plates.