Rpmi medium

CHO cells (CHO-K1) stably transfected with a calcium-sensitive bioluminescent fusion protein consisting of aequorin and green fluorescent protein (GFP) were kindly provided by Dr. Stefan Offermanns (Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany) and were cultured as described (30 (link)). These cells are indicated as CHO/G5A.
TFK-1-cells (ACC-344) originating from bile duct carcinoma (obtained from Leibniz DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in RPMI medium (Biochrom GmbH, Berlin, Germany), supplemented with 10% (v/v) fetal bovine serum (Gibco, Life Technologies, Darmstadt, Germany), and 160 μg/ml Gentamycin (Merck-Sigma-Aldrich, Munich, Germany).
Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ and used between passages 8 and 20.

Peripheral Blood Mononuclear Cells (PBMCs) from buffy coats were obtained from anonymous healthy blood donors donated by the Transfusion Centre of Madrid following national guidelines (n = 28) and collected in ethylenediaminetetraacetic acid (EDTA) using Ficoll (Ficoll-Paque TM PLUS) by density gradient centrifugation on blood collection day and used immediately. After isolation, PBMCs were resuspended in 10% RPMI medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin G, 100 µL/mL streptomycin sulfate and 1% l-glutamine) (Biochrom AG, Berlin, Germany). Interleukin-2 (60 U/mL) (rhIL-2; Bachem AG, Bubendorf, Switzerland) was added to PBMCs.

Human breast carcinoma cells MCF7 and MDA-MB-231, colon adenocarcinoma cells HCT116 (wt p53 and KO p53 cells) and wt p53 mouse melanoma cells B16 were cultivated in DMEM containing 4.5 g/l glucose (Biochrom, Berlin, Germany). All cells are from ATCC. Breast cancer NeuTL cells derived from tumors of transgenic FVB/N c-neu mice and 4T1 mouse breast carcinoma cells (ATCC) were cultivated in the RPMI medium containing 4.5 g/l glucose (Biochrom, Berlin, Germany). Media was supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin sulfate (Sigma). The RPMI medium was supplemented with sodium pyruvate (1 mM). Cells were kept at 37 °C under 5% CO₂. All cells were tested for mycoplasma contamination.

Cytokines were quantified in single-cell suspensions from the spleens and peritoneal macrophage cultures prepared 1 week after the last dose of DMBA. As there is almost no basal release of cytokines under in vitro conditions, lymphocytes (2.5 × 10⁵ cells/ml) were cultured in RPMI medium (Biochrom, Berlin, Germany) containing 10% (v/v) FCS (Biochrom) for 48 h with 1 μg/ml plate-bound anti-CD3e antibodies (eBioscience) for overall T-cell receptor stimulation. Macrophages (1.5x10⁵ cells/ml) were stimulated with 1 μg/ml LPS (Sigma-Aldrich) (4 h) and 5 mM ATP (InvivoGen, Toulouse, France) (1 h) in RPMI medium containing 10% (v/v) FCS. Cytokines were measured in the supernatants using standard sandwich ELISA procedures as previously described.^{48 (link)} Primary and biotin-labeled secondary antibodies for IL-1β, IL-6, TNFα, IL-2 and IFNγ were purchased from BD Biosciences (Heidelberg, Germany) and antibodies for IL-17 from R&D Systems (Wiesbaden, Germany). For quantification, recombinant cytokines were used as standard. Absorbance was measured at 492 nm. TGF-β1 was determined at the same time in the serum using an ELISA Kit (R&D Systems) according to the manufacturer's instructions.

A549 human lung epithelial cells (ATCC^® CCL-185) were grown in RPMI medium (Biochrom GmbH, Berlin, Germany) with 10% fetal calf serum (FCS) (Sigma, Taufkirchen, Germany) at 37°C until almost confluent and infected with bacteria at a multiplicity of infection (MOI) of 100. After 24 h, the supernatant was filtered (0.45 μm) and the lactate dehydrogenase (LDH) activity was determined by spectrophotometry (Cobas 8000, module 701; Roche, Grenzach-Wyhlen, Germany).

HL60 cells stably transfected with human FPR2/ALX (HL60-FPR2) have been described recently (23 (link)). These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM Hepes (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1 × Glutamax (Gibco) and in the presence of G418 (Biochrom) at a final concentration of 1 mg/ml. Neutrophils or HL60-FPR2 cells were stimulated with different bacterial culture filtrates (USA300, USA300Δαβδ, USA300ΔeapΔH1ΔH2::ermB, S. epidermidis RN1457) or FPR2 ligands (PSMα3, PSMε) as described in the figure legends. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes) and monitoring fluorescence with a FACSCalibur or a FACS Fortessa flow cytometer (BD Biosciences) as described recently (23 (link)).

Splenic B cells from MD4 mice were enriched using MACS technique as described above. B cells were cultured for 24 hours in RPMI medium (Biochrom, Berlin, Germany) with 10% FCS (GE Healthcare Life Sciences, Buckinghamshire, UK), 1% Pen/Strep (Gibco, Thermo Fisher Scientific, Waltham, USA) and 0,3mg/ml Glutamin (Biochrom) containing 1μg HEL (Sigma) for stimulation. Subsequently, cells were i.v. injected into WT mice, which were simultaneously orally gavaged with 1mg HEL or PBS. One day later mice were sacrificed and analysed via flow cytometry.