Penicillin streptomycin solution

Cell culture. Mouse macrophage RAW 264.7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in a DMEM/high-glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific Hyclone, USA) and 1% penicillin–streptomycin solution (Thermo Scientific HyClone, USA). Cells were grown to 70–80% confluency in a T-75 flask and cultured for 2 days at a density of 2 × 10⁵ cells/ml. Cells were then incubated at 37°C in a humidified 5% CO₂ incubator (Thermo Fisher Scientific, USA).
Cell viability. RAW 264.7 cells were seeded at a density of 3 × 10⁵ cells/ml with 100 μl/well in a 96-well plate. Yeast-fermented cabbages were treated at a concentration of 5 μg/ml for 24 h until the cells reached 70%–80%confluency. A total of 10 μl/well of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution was added to the mixture and incubated for 2 h. Later, 80 μl/well of the mixture was discarded, and 100 μl/well of dimethyl sulfoxide (DMSO) solution was added. After 30 min of incubation in a dark room, cell viability was detected with a microplate reader (Bio-Rad Inc., USA) at an absorbance of 595 nm.

Male mouse embryonic stem cells were grown on gelatin-coated plates at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukemia inhibitory factor. To induce conditional catalytic point mutation or knockout, PRC1^CPM or PRC1^CKO cells were treated with 800 nM 4-hydroxytamoxifen (OHT) for 72 h. Cells were regularly tested for the presence of mycoplasma.
Drosophila S2 (SG4) cells were grown adhesively at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 1x penicillin-streptomycin solution (Life Technologies) and 10% heat-inactivated fetal bovine serum (Labtech).
Human HEK293T cells were grown at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), and 0.5 mM beta-mercaptoethanol (Life Technologies).

293T cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution (Life Technologies). MDA-MB-231 cells were maintained in L-15 media with 10% FBS and 1% penicillin–streptomycin solution (Life Technologies). T47D and MCF7 cells were cultured in Roswell Park Memorial Institute medium-1640 (RPMI-1640) (Life Technologies) supplemented with 10% FBS and 1% penicillin–streptomycin solution (Life Technologies).

Peripheral blood from healthy volunteers were drawn in vacutainer EDTA tubes. Polymorphonuclear granulocytes were isolated using Polymorphprep™ (Axis-Shield, Oslo, Norway) and following the manufacturer’s instruction. Granulocytes (1x10⁶ cells) in 500 μl RPMI 1640 supplemented with L-glutamine and Penicillin/Streptomycin Solution (Thermo Fisher Scientific) were incubated at 37°C in the presence of N-Formyl-Met-Leu-Phe peptide, between 0.2–5.0 μM (Sigma-Aldrich, St Louis, MO, USA). At different time points, cells were removed by centrifugation and BSSL concentration in the culture media was determined by ELISA.
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using Ficoll-Paque PLUS solution (Cytiva, Uppsala, Sweden) according to manufacturer’s instruction. The PBMCs were suspended in RPMI 1640 supplemented with L-glutamine and Penicillin/Streptomycin Solution (Thermo Fisher Scientific). In cases where the PBMCs were intended for further sub-fractionation (monocyte isolation), the cells were suspended in PBS-EDTA. Monocytes were isolated from the PBMC fraction by negative selection using the monocyte isolation kit II (Miltenyi Biotech, Lund, Sweden) according to manufacturer’s instruction. The monocytes were suspended in RPMI 1640 supplemented with L-glutamine and Penicillin/Streptomycin Solution (Thermo Fisher Scientific).

The OvCa cell lines, OVCAR-3, and SW 626 were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). Following to the ATCC cell culture method with a small modification, OVCAR-3 cells were grown in RPMI media supplemented with 20% Fetal Bovine Serum (FBS) (Fisher scientific, PA, USA), 0.01 mg/ml bovine insulin, 1% of Non-essential amino acid solution and 1% mL of penicillin/streptomycin solution (Fisher Scientific, PA, USA). However, SW 626 cells were grown in L-15 media (Fisher Scientific, PA, USA), supplemented with 10% FBS, and penicillin/streptomycin solution. All cell cultures were maintained in a humidified incubator at 37 °C and 5% CO₂.