Leibovitz s l 15 cell culture medium

Sea cucumbers were sterilized in 7% benzalkonium bromide and 75% ethanol for approximately 2 min. Subsequently, the sea cucumbers were dissected using an aseptic surgery technique as previously described56 . The coelomic fluids were collected and mixed with an equal volume of anticoagulant solution (0.02 M EGTA, 0.48 M NaCl, 0.019 M KCl, and 0.068 M Tris-HCl, pH 7.6). The cell suspension was filtered through a 100-μm nylon mesh to remove large tissue debris and centrifuged at 800 x g for 10 min at 16 °C. The cells were washed twice with isotonic buffer (0.001 M EGTA, 0.53 M NaCl, and 0.01 M Tris-HCl, pH 7.6) and re-suspended in Leibovitz’s L-15 cell culture medium (Invitrogen, USA) supplemented with penicillin (100 U mL-1), streptomycin sulfate (100 μg mL-1), and NaCl (0.39 M) to adjust the osmotic pressure. Next, 1000-μL aliquots of cell suspension were dispensed into 24-well microplates and cultured for 6 h at 16 °C in a black room. For the LPS exposure, the primary cultured coelomocytes were exposed to 1 mg mL⁻¹ of LPS for 0 h and 6 h. Following the LPS exposure, the cells were washed in PBS, centrifuged at 800 × g and 4 °C for 5 min, and stored at –80 °C for the subsequent expression analysis.

CRC cell lines SW620 (ATCC, #CCL-227), SW480 (ATCC, #CCL-228), HCT116 (ATCC, #CCL-247), and HT29 (ATCC, #HTB-38) were purchased from American Type Culture Collection. SW620 and SW480 cells were cultured in Leibovitz's L-15 cell culture medium (Gibco). HCT116 and HT29 cells were cultured in McCoy’s 5A cell culture medium (Gibco). Media was supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) PenStrep, all purchased from Invitrogen. SW480 OxR, HCT116 OxR, and HT29 OxR cells were obtained from MD Anderson Cancer Center Characterized Cell Line Core, supplied and generated by the Dr. Lee Ellis laboratory. SW620 OxR cells were obtained from Dr. Mika Hosokawa at Kobe Pharmaceutical University in Japan. OxR derivative cell lines were cultured in the same medium as their parental counterparts. To prevent phenotypic drift of OxR lines, cells were used within six passages from the time they were received. To prevent chemotherapy-induced cytotoxicity in downstream experiments, oxaliplatin was not supplemented in OxR cell culture media. All cell lines were maintained in a humidified incubation chamber at 37°C and 5% CO₂. All cell lines were screened for mycoplasma contamination and tested negative.