1640 medium with stable glutamine

The leucocyte concentration including lymphocyte and neutrophil concentrations was very similar between dogs measured by a CBC. Heparinized blood was diluted to a ratio of 1:10 with Rosewell Park Memorial Institute (RPMI) 1640 medium with stable glutamine and 25 mM hepes (Biowest®, USA) supplemented with 60 μg/ml of penicillin, 100 μg/ml streptomycin (Life Technologies™, USA) and 10 % Fetal Bovine Serum Premium South America Origin (Biowest®, USA). Five hundred μl of heparinized blood was mixed with 4.5 ml of complete medium as described above per each well and incubated in 12-well flat bottom plastic culture plates 3596 (Costar® Corning, NY, USA). Three different treatment conditions were established: (i) medium alone; (ii) medium with L. infantum soluble antigen (LSA) at a concentration of 10 μg/ml provided by Dr. Cristina Riera (Facultat de Farmacia, Universitat de Barcelona); and (iii) medium with mitogen concanavalin A (ConA) (100 mg Medicago®, Sweden) at a concentration of 10 μg/ml. The plates were incubated for 5 days at 37 °C in 5 % of CO₂ air. Following incubation, blood was centrifuged at 300 g for 10 minutes and the supernatant was collected and stored at -80 °C until used.

The colorectal cancer-derived cell lines SW480 and RKO, harboring KRAS and BRAF mutations, respectively, were obtained from the American Type Culture Collection (Manassas, VI, USA). The noncancerous NCM460 cell line derived from normal colon epithelial mucosa was obtained from INCELL’s (San Antonio, TX, USA) [30 (link)]. The cells were grown at 37 °C under a humidified atmosphere containing 5% of CO₂. RKO cell lines were grown in Dulbecco’s Modified Eagle´s Medium High Glucose (Biowest, Nuaillé, France). Both the SW480 and NCM460 cell lines were grown in Roswell Park Memorial Institute 1640 medium with stable glutamine (Biowest, Nuaillé, France). Both mediums were supplemented with 10% fetal bovine serum (v/v) (Biowest, Nuaillé, France) and 1% penicillin/streptomycin (v/v) (Biowest, Nuaillé, France). The cells were subcultured once a week when 80% of confluence was reached and, when necessary, seeded in sterile test plates for the assays. The SW480 and RKO cells were seeded at 1 × 10⁵ cells/mL concentration and the NCM460 at 2 × 10⁵ cells/mL, except for some specific assays.

NCM460 and SW480 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with stable glutamine (Biowest, Nuaillé, France) supplemented with 1% penicillin-streptomycin (Biowest, Nuaillé, France) and 10% heat-inactivated fetal bovine serum (FBS; Gibco, Invitrogen, Waltham, Massachusetts, USA); RKO cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) High Glucose (Biowest, Nuaillé, France) supplemented with 1% penicillin-streptomycin (Biowest, Nuaillé, France) and 10% heat-inactivated FBS (Gibco, Invitrogen, Waltham, MA, USA). HCT116 cells were grown in McCoy’s 5A Medium (Biowest, Nuaillé, France) supplemented with 1% penicillin-streptomycin (Biowest) and 10% heat-inactivated FBS (Gibco, Invitrogen, Nuaillé, France). All cell lines were plated onto 25 cm³ tissue culture flasks, maintained in a humidified incubator with 5% CO₂ at 37 °C.