Bbl l wenstein jensen medium

For peptide synthesis, amino acid derivatives were obtained from Iris Biotech (Marktredwitz, Germany). Reagents such as N,N’-diisopropylcarbodiimide (DIC), triisopropylsilane (TIS), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), NH₄OAc, palmitic acid, 6-maleimidohexanoic acid, acetic anhydride (Ac₂O), N,N-diisopropylethylamine (DIEA), 2,2,2-trifluoroethanol (TFE), and 5(6)-carboxyfluorescein (Cf) were purchased from Sigma-Aldrich (Budapest, Hungary). Fmoc-Rink Amide MBHA resin was obtained from Merck. Trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), dichloromethane (DCM), 2-ethoxyethanol (Cellosolv), diethyl ether, and acetonitrile (AcN) were from VWR (Budapest, Hungary).
For the in vitro assays, RPMI-1640 medium, fetal calf serum (FCS), 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), concanavalin A (ConA), Mowiol^® 4–88, trypan blue, and trypsin were obtained from Sigma-Aldrich (Budapest, Hungary). Macrophage colony-stimulating factor (M-CSF) was from Shenandoah Biotechnology (Warwick, PA, USA). BBL Löwenstein–Jensen Medium was ordered from Becton Dickinson (Környe, Hungary), HPMI buffer (9 mM glucose, 10 mM NaHCO₃, 119 mM NaCl, 9 mM HEPES, 5 mM KCl, 0.85 mM MgCl₂, 0.053 mM CaCl₂, 5 mM Na₂HPO₄ × 2H₂O, pH = 7.4) was prepared in-house using components obtained from Sigma-Aldrich (Budapest, Hungary).

MonoMac-6 human monocytes (2 × 10⁵ cells/1 mL medium/well) were cultured in RPMI-1640 medium containing 10 % FCS, in a 24-well plate, 24 h prior to the experiment. Adherent cells were infected with Mycobacterium tuberculosis H₃₇Rv, at a multiplicity of infection (MOI) of 10. Nonphagocytosed extracellular bacteria were removed by washing the culture three times with serum-free RPMI. After 1 day of incubation, infected cells were treated with INH-conjugated Penetratin and Transportan 5, 10 and 50 μM final concentrations. For comparison, INH was also assayed at 100 μM concentration. As control, infected cells were treated with culture media. After 3 days, the treatment was repeated with fresh solutions of the compounds, and cells were incubated for an additional 3 days. After 3 washing steps, infected cells were lysed with 2.5 % sodium dodecyl sulphate (SDS) solution, and 100 μL lysate was transferred to L-J medium (BBL Löwenstein–Jensen medium, Beckton Dickinson). Colony-forming units (CFU) were enumerated after 4 weeks of incubation, and the number of bacteria was calculated by using a standard dilution series of M. tuberculosis [60 (link)].

Amino acid derivatives and resins were obtained from Iris Biotech (Marktredwitz, Germany). Reagents, such as N,N′-diisopropylcarbodiimide (DIC), triisopropylsilane (TIS), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), Isoniazid (INH), glyoxylic acid and 5(6)-carboxyfluorescein (Cf) were purchased from Sigma (Budapest, Hungary). Trifluoroacetic acid (TFA) and acetonitrile (AcN) were from VWR (Budapest, Hungary). N,N-dimethylformamide (DMF), dichlormethane (DCM), diethyl ether, and ethanol were purchased from Reanal (Budapest, Hungary)
For the in vitro assays, RPMI-1640 medium, fetal calf serum (FCS), and trypan blue were obtained from Sigma (Budapest, Hungary), while DMEM medium and 2 mM l-glutamine were from Lonza (Basel, Switzerland). Trypsin, nonessential amino acids, and penicillin-streptomycin were from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). HPMI buffer (9 mM glucose, 10 mM NaHCO₃, 119 mM NaCl, 9 mM HEPES, 5 mM KCl, 0.85 mM MgCl₂, 0.053 mM CaCl₂, 5 mM Na₂HPO₄ × 2H₂O, pH = 7.4) was prepared in-house using components obtained from Sigma (Budapest, Hungary). BBL Löwenstein-Jensen medium was from Becton Dickinson (Környe, Hungary).