Rpmi 1640 culture medium

Blood (6 ml) was collected in ethylenediamine tetra-acetic acid tubes from each WZSP maintained by Grand Life Science and Technology Ltd., Beijing, China. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation in lymphocyte separation medium (Tianjin HAO YANG Biological Manufacture Co., Ltd., Tianjin, China). After three washes in phosphate-buffered saline (PBS), half of the PBMCs were cryopreserved in RPMI 1640 culture medium (Gibco, Carlsbad, CA, USA) supplemented with 40% fetal bovine serum and 10% dimethyl sulfoxide. The rest of the PBMCs were mitogenically stimulated in the following medium: RPMI 1640 culture medium supplemented with 10% fetal bovine serum, 2.5 μg of phytohemagglutinin (PHA) per ml, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Carlsbad, CA, USA).

The proliferative activity of PBMCs stimulated by concanavalin A (ConA) and Phorbol 12-myristate 13-acetate (PMA) was measured as described previously [42 (link)]. Isolated chicken PBMCs were obtained using a peripheral chicken blood mononuclear cell isolate kit (TBD, Tianjin, China). PBMCs were diluted to 1 × 10⁶ cells/mL with RPMI 1640 culture medium containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and penicillin-streptomycin (Wisent, Saint-Jean-Baptiste, QC, Canada). Then, 100 μL PBMCs were added to each well of the 96-well plates. Next, 5 μg/mL ConA and 100 ng/mL PMA were added to each well and maintained at 37 °C in a cell incubator for 48 h. Subsequently, 10 μL CCK-8 was added for 4 h, and absorbance was measured at 450 nm.
The proliferative activity of PBMCs stimulated with Lipopolysaccharide (LPS) was measured as described previously [43 (link)]. PBMC was diluted to 1 × 10⁶ cells/mL with RPMI 1640 culture medium containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and penicillin-streptomycin (Wisent, Saint-Jean-Baptiste, QC, Canada). Subsequently, 100 μL of this solution were added to a 96-well plate, along with LPS at a final concentration of 1 μg/mL per well, and maintained at 37 °C for 72 h in a cell culture incubator. Next, 10 μL CCK-8 were added for 4 h, and absorbance was measured at 450 nm.

THP-1 cells were cultured in RPMI 1640 culture medium (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS, Corning), 2 mmol/L of glutamine (Gibco) and the antibiotics 100 U/mL penicillin and 100 µg/mL streptomycin (Pen Strep, Gibco), based on a previously described protocol^{20 (link),61 (link)}. Briefly, cells were seeded and differentiated using 85 nM Phorbol 12-Myristate 13-Acetate (PMA, Sigma) for 3 days, media was replaced, and cells were allowed to rest for 5 additional days. The medium was replaced and cells were subjected to treatments with 100 µg/mL of the peptides 65-79*LE, 65-79*SE or 65-79*PE with or without recombinant human IFN-γ (10 ng/mL).
RAW 264.7 cells were cultured in DMEM culture medium (Gibco) supplemented with 10% FBS (Corning) and 100 U/mL penicillin and 100 µg/mL streptomycin (Pen Strep, Gibco). Cells were plated and treated with 65-79*LE, 65-79*SE or 65-79*PE (100 µg/mL) with or without recombinant mouse IFN-γ (5 ng/mL).

Primary lymphocytes cultures were obtained from heparinised whole blood samples processed within 24 h from collection. At least six cultures (two for each analysis performed) were assessed from each subject. 0.3 ml of blood were added to 4.7 ml of RPMI-1640 culture medium (Invitrogen, Milano, Italy) supplemented with 20% FBS, 1% antibiotics and antimycotics and 1.5% phytohemagglutinin (Invitrogen, Milano, Italy). Cultures were kept at 37° for a variable time depending on the specific analysis requirements. For γH2AX analysis, cultures were immediately harvested in order to avoid lymphocytes stimulation, while for GSTO1 immunostaining they were harvested after 24 h proliferation. For assessment of MN induction in mononucleated cells, cultures where harvested after 72 h of incubation as described in the appropriate subsection (see below). In all the analyses performed, lymphocytes harvesting was performed using the same protocol described elsewhere [31 (link)]. Briefly, cells were initially precipitated by a 5 min centrifugation at 2100 rpm. After replacing the medium with 5 ml of hypotonic solution (KCl, 0.075 M), cells were pre-fixed in 0.4 ml of 5:3 acetic acid:methanol, re-centrifuged and the obtained pellet was resuspended in pure methanol for sample storage at −18°C.

The colorectal cancer cell line CT26 and human colorectal cancer cell line HCT116 were purchased from the cell bank of the Chinese Academy of Sciences. The two colorectal cancer cell lines were both maintained in RPMI 1640 culture medium (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Invitrogen), 100 units/ml penicillin and 100 mg/ml streptomycin in a humidified incubator under 95% air and 5% CO₂ at 37 °C.

LoVo cells were maintained in RPMI-1640 culture medium (Gibco Invitrogen Corporation, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Beyotime Institute of Biotechnology, Haimen, China) and 2 mM L-glutamine (Amresco LLC, Solon, OH, USA). The cells were grown in monolayer cultures with 5% CO₂ in a humidified 37°C incubator. Every 2–3 days, the cells were subcultured. When the cells reached the logarithmic growth phase, 0.25% trypsin (Amresco LLC) was applied for 1–3 min. The cells were resuspended in RPMI-1640 containing 10% FBS at a cell concentration of 1×10⁴ cells/ml.