Raw 264.7 macrophage cell line

Murine RAW264.7 macrophage cell line (ATCC) was maintained according to ATCC’s recommendations in DMEM-GlutaMAX media containing 10% FBS and penicillin-streptomycin (100 IU/ml and 100 mg/ml). For generation of murine bone marrow-derived dendritic cells, bone marrow cells were sterilely harvested from the tibia and femur, red blood cell lysed using sterile Red Blood Cell Lysing Buffer Hybri-Max (Sigma) and frozen in FBS containing 10% DMSO (Sigma) at −80 °C. Bone marrow cells were thawed, rinsed to remove DMSO and resuspended at a concentration of 5 × 10⁵ cells/mL in culture media RPMI 1640-GlutaMax containing 5% FBS, 50uM 2-mercaptoethanol, penicillin-streptomycin (100 IU/ml and 100 mg/ml) and 20 ng/ml GM-CSF (BioLegend). After day 3, all non-adherent cells were removed, and adherent dendritic cell clusters were cultured in the fresh medium for another 6 days before use. Every other day half of the medium was replaced with fresh culture medium.

The RAW264.7 macrophage cell line was obtained from ATCC. The ability of compounds 1–7 to inhibit the expression of iNOS (inducible nitric oxide synthetase) and COX-2 (cyclooxygenase-2) pro-inflammatory proteins in LPS-induced RAW264.7 macrophage cells was assessed to determine their in vitro anti-inflammatory activities. A detailed description of the method used for this evaluation was provided in previous publications [2 (link)].

B. abortus strain 2308, B. suis strain 1330, B. melitensis strain 16M, and avirulent strain M5 were obtained from the Chinese Veterinary Culture Collection Center (Beijing, China). E. coli strains were routinely cultured aerobically at 37°C in lysogeny broth (LB) and on LB agar plates. The Brucella strain and its derivatives were routinely grown on TSB (Difco, Franklin Lakes, NJ, USA) or TSA (Difco) at 37°C under an atmosphere of 5% CO₂. When needed, 50 µg/mL of kanamycin or 20 µg/mL of chloramphenicol was added to the broth. The RAW264.7 macrophage cell line (ATCC TIB-71, Manassas, VA, USA) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco) at 37°C in a 5% CO₂ atmosphere.

To validate the up-regulation of MMP-2 and MMP-9 during Mtb infection, we constructed reporter cell lines that have promoters of Mmp-2 and Mmp-9 upstream of GFP and luciferase encoding cassettes. The luminescent signal was detected and quantified by the IVIS imaging instrument. The primers sequences for Mmp-2 and Mmp-9 promoter’s region were designed using PrimerPremier5 (Table 1).
Mouse genome DNA was used as template to amplify the sequences, which were inserted into plasmid pGreen-Fire. Vectors with genes of interest were transferred into RAW 264.7 macrophage cell line (ATCC TIB-71) using a lentivirus infection system. Single colonies were picked and validated for expression levels of GFP and luciferase. We used a non-infectious Mtb trehalose dimycolate (TDM) granuloma model to test whether these stable cell lines can express GFP and luciferase [29 (link), 31 (link)]. TDM coated beads were suspended in Matrigel, mixed with the reporter RAW cell lines, and inoculated subcutaneously in the scruff of a mouse. The mice were anesthetized at certain time points, injected with luciferase substrate and imaged with a IVIS machine (Caliper Lifescience).

White mulberry leaves (M. alba L.) from Sakon Nakhon and Buriram cultivars were collected during May–June in Chiang Mai province (northern Thailand: 19°00′ N, 99°00′ E) under the supervision of the Queen Sirikit Department of Sericulture. The leaves were dried in an oven at 60 °C for 48–72 h and blended into powder with a blender. The samples were kept in airtight plastic bags for around 1–2 days prior to extraction.
RAW 264.7 macrophage cell line originated from mice (Mus musculus) and was purchased from ATCC with the accession number ATCC-TIB-71.