Non enzymatic cell dissociation solution

Tumors were mechanically fragmented by cutting with sterile scissors in a DMEM cell culture medium. The cell-containing DMEM medium was filtered through 40 μm cell strainers. After harvesting by means of centrifugation at 300× g for 3 min, cells were incubated in the non-enzymatic cell dissociation solution (Corning) at 37 °C for 10 min. Then, cells were harvested and re-suspended in the binding buffer for the aptamer or antibody binding assay as described above.

Mice were intraperitoneally treated with 3% thioglycolate for 4 days, and peritoneal lavage was collected by injecting ice-cold PBS into the peritoneum. Peritoneal cells were washed, counted and resuspended in IMDM media containing 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin and 0.1 mM β-mercaptoethanol. After waiting for at least 2 h, enabling adherence to a well plate, non-adherent cells were removed by changing the medium. The adherent peritoneal macrophages were pretreated with the indicated herbal compounds for 1 h and activated with 200 ng/mL LPS cells for 8 h. Bone marrow-derived macrophages (BMDMs) were prepared as described previously [37 (link)]. Briefly, bone marrow cells were isolated from femurs and tibias and cultured for 7 days in a Petri dish in the presence of 20 ng/mL M-CSF. BMDMs were harvested by adding non-enzymatic cell dissociation solution (Corning, Tewksbury, MA, USA) to the Petri dish and seeding them into a well-plate. RAW264.7 cells were provided by Hyung-Joo Kwon (Hallym University, Chuncheon, Korea) and cultured in DMEM supplemented with 10% FBS. RAW264.7 cells were transfected with MSCV-Akt(DD)-IRES-Thy1.1 [AKT^DD], MSCV-IKK2(SSEE)-IRES-Thy1.1 [IKK2^SSEE], or control vector [EV] using the FuGene^® HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions.

The TNBC cell lines were kind gifts from Dr. Jenny C. Chang’s lab at the Houston Methodist Academic Institute. The other cells were purchased from American Type Culture Collection (ATCC, Manassas, VI, USA), cultured, and stored in our laboratory. Cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Corning, Corning, NY, USA) supplemented with 10% FBS (Corning), 100 units/mL penicillin (Gibco, Waltham, MA, USA), and 100 μg/mL streptomycin (Gibco). For cell passage, 0.25% trypsin with ethylenediaminetetraacetic acid (Corning) and non-enzymatic cell dissociation solution (Corning) were used. All cells were grown at 37 °C in 95% air/5% CO₂. All experiments were performed with mycoplasma-free cells as tested with e-Myco™ plus mycoplasma PCR detection kit (iNtRON Biotechnology, Seoul, South Korea).