Rpmi 1640

Roswell Park Memorial Institute (culture medium) (RPMI1640), Fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) were purchased from Himedia, India. The general chemicals were also purchased from Sigma or Merck, India. The rat pheochromocytoma (PC12) cells line used in the present study were obtained from the National Center for Cell Science, Pune, India.

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.

THP1 cells were cultured in RPMI-1640 (HiMedia Laboratories, Mumbai, India) media supplemented with 10% fetal bovine serum (FBS) and 1 mM of sodium pyruvate (HiMedia Laboratories, Mumbai, India). HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 1 mM of sodium pyruvate. For differentiation, THP1 cells were treated with 100 nM of PMA for 24 h and rested for 48 h before the next manipulation. The identity of both cell lines was confirmed by short tandem repeat (STR) PCR analysis, and the cell lines were maintained mycoplasma free by routine monitoring. LPS treatment of macrophages was performed with 10 ng/ml of LPS for 6 h. TLR4 signaling was inhibited by treatment with 10 µM of CLI095 before infection or LPS treatment. For ERK inhibition, THP1 macrophages were treated with 2 µM of U0126 for 6 h. To inhibit serine biosynthesis, macrophages were treated with 10 µM of NCT-503 for 6 h, and 10 µM of PHGDH-inactive compound was used as the negative control.