Roswell park memorial institute (rpmi)

Peritoneal exudates (PE) cells were isolated and plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (RPMI+; all from Biochrom AG, Berlin, Germany). After 2 h of incubation, purified PM were washed, detached by 2mM PBS/EDTA, counted and plated in RPMI+ medium (2 × 10⁵ cells/96 well). Secretion of NO by PM was measured in supernatants after over-night stimulation with 100 ng/ml lipopolysaccharide LPS (L2654; Sigma-Aldrich) in Griess assay, as described previously (20 (link)).
For the assessment of metabolic activation, PM were purified from freshly isolated PE with EasySep™ Mouse Monocyte Isolation Kit (Stemcell Technologies, Vancouver, Canada). Cells were plated (10⁵ in 100 μl RPMI+ medium) and ATP amount was determined by CellTiter-Glo® and Glomax luminometer (Promega, Madison, Wisconsin, USA).
Autophagy in PM was measured in freshly isolated PE by flow cytometry according to the kit manual (Cyto-ID® autophagy detection kit, Enzo Life Sciences, Farmingdale, New York, USA) in parallel with staining of surface markers.

Cell lines were regularly tested for the absence of contamination/mycoplasma and STR-typed. Cell lines were cultured in RPMI (Biochrom) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% penicillin/streptomycin (Gibco, ThermoFisher Scientific), and 1% L-glutamine (Gibco, ThermoFisher Scientific).

Cell lines were maintained as early passages of subconfluent cultures in RPMI or DMEM (Biochrom AG, Berlin, Germany) at 5% or 10% CO2, respectively, supplemented with 10% FBS (Biochrom AG) and 1% NEAA (Biochrom AG). RT112 and 647V from the Leibniz institute German collection of microorganisms and cell cultures (Braunschweig, Germany), UMUC3, J82 and T24 were obtained from the American type culture collection (Manassas, VA, USA), 639 V and VmCUB1 were a kind gift from Professor Dr WA Schulz (Heinrich-Heine-University, Düsseldorf, Germany) and 253J were kindly provided by Professor Dr G. Unteregger (University of Saarland, Homburg/Saar, Germany). Cell lines were authenticated by short tandem repeat profiling and tested for mycoplasma using PCR as described previously [24 (link),25 (link)].

A total of 2 × 10⁶ PBMC per well were seeded in sterile cell culture 12-well plates and incubated for 12 h in a humidified atmosphere at 37 °C (5 % CO₂ in air) in 1 ml culture medium [(Roswell Park Memorial Institute: RPMI, Biochrom AG, Berlin, Germany) supplemented with 10 % heat-inactivated foetal calf serum (FCS; PAA Laboratories GmbH, Pasching, Austria) and penicillin 100 U/ml, streptomycin 0.1 mg/ml (PAA)] (medium only), supplemented with 1 μg/ml lipopolysaccharide (LPS; #L2755, Sigma-Aldrich, Munich, Germany) (LPS-stimulated), or in medium supplemented with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (1.34 μM) (Cell stimulation cocktail 500×, eBioscience, Frankfurt, Germany) (PMA/ionomycin-stimulated). Rather high concentrations of PMA/ionomycin were chosen after preliminary experiments (data not shown) to obtain positive controls of cytokine secretion for each individual.
After incubation, cell-free supernatants were obtained and stored at −80 °C until further analysis. Thawing was performed at 37 °C immediately prior to the cytokine determinations.
Cells in medium only were used to evaluate spontaneous cytokine release. Lipopolysaccharide stimulation was used to evaluate TLR4-response [35 (link)] and PMA/ionomycin stimulation to assess cytokine induction by non-specific stimulation [36 (link)–38 (link)] as positive controls.

K-562 cells were acquired from Sigma-Aldrich and cultured at 37 °C with 5% CO₂ in RPMI (Biochrom) supplemented with 10% FBS (Gibco), 1% L-glutamine (Biochrom), and 1% Penicillin/Streptomycin (Biochrom).
Immunomagnetic enrichment of HSPCs was performed using magnetic-activated cell sorting system (CliniMACS System, Miltenyi Biotec), according to the manufacturer’s instructions. CD34⁺ HSPCs were then cultured at 37 °C with 5% CO₂ in StemMACS HSC Expansion Media (Miltenyi Biotec) supplemented with human cytokines (Miltenyi Biotec): SCF (100 ng/ml), TPO (20 ng/ml), and Flt3-L (100 ng/ml).

The human epithelial CRC cell line RKO (ATCC CRL 2577), normal colon cell line CCD-841-CoN (ATCC CRL 1790) and CRC cell line Caco-2 (ATCC HTB-37) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Biochrom). The human CRC cell lines HCT 116 (ATCC CCL-247) and HCT-15 (ATCC CCL-225) were grown in Roswell Park Memorial Institute Medium 1640 (RPMI, Biochrom) supplemented with 10% (v/v) fetal bovine serum (FBS, Biochrom) and 1% (v/v) penicillin-streptomycin (Biochrom). Cells were maintained at 37 °C with 5% CO₂.

The human GBM cell lines U87MG and U373MG (kindly provided by Dr. Joseph Costello, University of California San Francisco, CA, USA), SNB19 (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), U251MG, A172, LN229 (ATCC, American Type Culture Collection, Manassas, VA, USA), and the GBM patient‐derived cultures GBML18 (previously established in our group [22 (link)]), GBML24, GBML45, and GBML95 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biochrom GmbH, Berlin, Germany), supplemented with 10% fetal bovine serum (FBS, Biochrom GmbH). The GBM patient‐derived culture GBML42 (established in our group, as previously described [23 ]) was maintained in Roswell Park Memorial Institute (RPMI, Biochrom GmbH) 1640 medium, supplemented with 10% FBS. Human embryonic kidney HEK293T cells (kindly provided by Dr. Andreia Neves‐Carvalho, ICVS/University of Minho, Portugal) used for lentiviral packaging were cultured in DMEM supplemented with 10% FBS. Complete medium is defined as DMEM or RPMI (according to the cell culture) supplemented with 10% FBS. All cells were maintained in a humidified atmosphere, with 5% (v/v) CO₂ and at 37 °C, unless otherwise stated. GBM cell lines were authenticated by short tandem repeat profiling. All cultures were tested for mycoplasma contamination every month.