Rpmi 1640 with stable glutamine

Prior to in vitro treatment, PBMCs were labelled with the CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, MA), as described by Reutner et al. (2012) (47 (link)). Subsequently, labelled PBMCs [2 × 10⁵/well] were cultivated in cell culture medium (RPMI1640 with stable glutamine [PAN-Biotech], supplemented with 10% [v/v] heat-inactivated fetal calf serum [FCS, Gibco™, Thermo Fisher Scientific]), with or without Pam3Cys-SKKKK [10 µM] or R848 [2.5 µg/ml] stimulation and in the presence or absence of DON [0.1-1.6 µM], DOM-1 [1.6 µM], ZEN [2.5-40 µM], or HZEN [40 µM] for 4 days at 37°C. Per condition, at least four wells were prepared (quadruplicates). Subsequently, wells treated with the same conditions were pooled and centrifuged (350g, 10 minutes room temperature). Supernatants were collected and stored at -20°C until use in ELISA. Cell pellets were resuspended and subjected to B-cell phenotyping and proliferation analysis by flow cytometry (FCM) as outlined below.

The human leukemia cell line K562 (26 (link)) and isolated porcine PBMC were propagated in RPMI 1640 with stable glutamine (PAN Biotech) supplemented with 10% (v/v) heat-inactivated FCS (PAA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (PAA). Cell culture medium for sorted cell subsets was additionally supplemented with 1 mM sodium pyruvate (PAA), non-essential amino acids (PAA), and 50 μM 2-mercaptoethanol (Sigma-Aldrich).

For the experiments, the following human colorectal cancer cell lines were used: DLD1, LoVo, LS-174T, SW620, SW480, SW837, HT29, and HCT116. The origins of these cell lines are given in Supplementary Table 1. All cell lines were genotyped using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) and were regularly tested negative for mycoplasma contamination.
Except for SW620 all cell lines were cultured in RPMI 1640 with stable glutamine and 2.0 g/L NaHCO3 (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech) and 1% penicillin‒streptomycin (P/S; PAN-Biotech). The cell line SW620 was cultured in DMEM (Gibco/Life Technologies) supplemented with 10% FBS and 1% P/S. The cells were cultured in a humidified atmosphere at 37 °C and 5% CO₂. For the collection of cell pellets, cells were seeded at specific cell numbers (1.5 × 10⁶ for HCT116 and HT29; 3.0 × 10⁶ for DLD1 and LoVo; 2.5 × 10⁶ for LS-174T and SW480; 4.0 × 10⁶ for SW837; 7.0 × 10⁶ for SW620) and collected after 48 h.