Raw264

The mouse macrophage cell line RAW 264.7 (ATCC TIB-71) was maintained in DMEM (Life Technologies) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Fisher Scientific, Pittsburg, PA, USA) at a density not exceeding 5 × 10⁵ cells/mL. Passages were performed every 3–4 days in 57 cm² cell-culture dishes (Nalge Nunc International, Rochester, NY, USA) maintained at 37 °C in a humidified 5% CO₂ Thermo Forma Series II incubator (Fisher Scientific).

The cell lines used in this study were purchased from the American Type Culture Collection (ATCC Manassas, VA, USA) and included the human colon carcinoma cell line HCT116, human colon carcinoma cell line SW620, human breast cancer cell line MCF-7, human embryonic kidney cell line HEK293T, mouse breast cancer cell line 4T1, mouse colon carcinoma cell line CT26, mouse melanoma cell line B16, mouse lymphoma cell line YAC-1, and murine macrophage cell line RAW264.7. The HCT116, SW620, 4T1, B16, and YAC-1 cell lines were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FBS (FBS; Gibco) and 1% penicillin/streptomycin. MCF-7, HEK293T, CT26, and RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% inactivated FBS and 1% penicillin/streptomycin. Stable TRAILR-KO cells (4T1-TRAILR-KO and CT26-TRAILR-KO) were generated using CRISPR/Cas9. All cell lines were cultured in an incubator at 37 ℃ and 5% CO₂. All cell lines were identified by short tandem repeat mapping at the start of this project. All cell lines were passaged for 2 months to ensure authenticity of cell line identity. All cells were tested for mycoplasma contamination, and all cell lines in this study were confirmed to have received treatment for mycoplasma contamination.

Murine macrophages (RAW 264.7, obtained from China Cell Bank) were cultured in DMEM medium at 37 °C in a 5% CO₂ humidified incubator [9 (link)]. A density of 1 × 10⁵ cells/mL was seeded in each well of a 6-well culture plate for 24 h. The culture supernatants of three different strains (OD₆₀₀ of 1.0 units) (L. gasseri FWJL-4, L. plantarum Fjias-5 and L. rhamnosus FSJ-13) and positive control (LGG, OD₆₀₀ of 1.0 units) were administrated to each well for 1 h, and then lipopolysaccharides (LPS, 1 μg/mL) were added for 20 h. The cell supernatants were obtained by centrifugation and filtration using a 0.22-μm membrane. According to the manufacturer’s protocol, Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA) was used to exact total RNA from RAW 264.7 cells. The mRNA expression levels of interleukin-10 (IL-10), IL-6, IL-1β and tumor necrosis factor (TNFα) were determined using the CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). The relative mRNA expression levels were normalized using the mRNA levels of β-actin. Primers used for real-time quantitative PCR (RT-qPCR) were indicated in Table 1.

The murine macrophage cell line RAW264.7 (ATCC number: TIB-71) was maintained at 37°C in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone, Logan, UT, USA) and antibiotics in a 5% CO₂ atmosphere. The human monocyte cell line THP-1 (ATCC number: TIB-202) was cultured in RPMI1640 (Invitrogen) supplemented with 10% FBS (HyClone). Prior to Mtb infection, THP-1 cells were treated with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow differentiation into macrophages. BMDM of > 95% purity were obtained from BALB/c as described previously [26 (link)]. Macrophage stimulation with Mtb strains was performed according to previous reports [16 (link), 27 (link)]. Briefly, macrophages were infected with clinical isolates of Mtb or H37Rv at multiplicity of infection (MOI) of 10:1. Four hours after infection, macrophages were washed twice with prewarmed serum-free RPMI1640 or DMEM to remove unbound bacilli, and were further cultured in serum-supplemented RPMI1640 or DMEM for another 4 hours. No toxicity was observed in Mtb-infected macrophages.