Pancoll

Blood was collected into sodium citrate collection tubes and was subjected to isolation of granulocyte and PBMC fractions within 1 h of draw. PBMCs and granulocytes were isolated using a density gradient 1.077 g/ml centrifugation, with the aid of Pancoll (PAN Biotech). Granulocytes were recovered from the corresponding erythrocyte fraction of the Pancoll gradient as previously described (19 (link)). Eosinophils were further purified via negative magnetic sorting using the Human Eosinophil Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. The purity of the isolated cells was examined by flow cytometry based on FSC and SSC parameters and low CD16 immunoreactivity. The eosinophil preparations were routinely ≥94% pure.

PBMCs were isolated from EDTA anticoagulated blood patient samples through Pancoll (Pan-Biotech) density gradient centrfugation. For best data comparability, all PBMC samples from one patient during therapy were frozen and thawed simultaneously at the day of experiment. PBMCs were thawed in complete medium (RPMI 1640 with 10% fetal bovine serum, 1% penicillin-streptomycin and 1.5% 1 M HEPES (all Thermo Fisher, Germany)) and incubated for 15–30 min at 37 °C in complete medium containing 50 U ml⁻¹ benzonase (Sigma, Germany) before processing.

For isolation of peripheral blood mononuclear cells (PBMC), whole blood was separated by density gradient centrifugation using Pancoll (PAN-Biotech, Germany). PBMC were collected and washed with PBS-EDTA (1 mM; used for all analyses). Cell count was determined using Neubauer improved haemocytometer. Single cell suspensions from spleen and lymph nodes were prepared by mechanically disrupting tissue with a sieve. Lymphocytes from liver were isolated following a modified protocol previously described (45 (link)). In brief, liver samples were perfused with ice-cold PBS-EDTA. Perfused regions were minced with sterile scissors, resuspended in PBS-EDTA supplemented with 100 μM CaCl₂, and digested with Collagenase D (1 mg/ml; Sigma-Aldrich) for 40 min at 37°C. Remaining tissue was removed by short centrifugation. Cell pellet was resuspended in PBS-EDTA and used for flow cytometry. Lymphocytes from lung tissue were isolated by mincing non-perfused lung tissue, followed by enzymatic digestion as described for liver samples. Lung tissue was additionally mashed through a cell strainer with the plunger of a syringe after digestion. Unless otherwise stated, cells were cultured in Ham's F12/IMDM (1:1), supplemented with 10% fetal calf serum (FCS), 2-mercaptoethanol (50 μM), 100 U/ml penicillin, and 100 μg/ml streptomycin.

To isolate T cells from human blood, leukocyte reduction chambers (provided by Transfusion Medicine, University Clinics Regensburg) were used. Leukocytes were first diluted three times with Dulbecco’s Balanced Salt Solution (DPBS) (#14190-094; Gibco) and the resulting blood and DPBS mixture was split into two fractions and underlayed with an equal amount of Pancoll (#P04-601000; PAN biotech). Samples were centrifuged at 1,000 × g for 20 min at room temperature (RT), with acceleration set to 4 and brake to 0. The PBMC layer was isolated and washed twice by centrifugation steps. Cells were pre-enriched with biotinylated anti-human CD8 (clone HIT8a; Biolegend), followed by column-based magnetic separation with anti-biotin ultrapure microbeads (#130-105-637; PAN biotech) following the manufacturer’s protocol.

Gastric cancer cell lines NUGC4 (JCRB0834) and NCI-N87 (ATCC^®CRL-5822) were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin, and maintained at 37 °C in a humidified atmosphere (5% CO₂). Cells were tested routinely for mycoplasma contamination. H. pylori strain G27 [31 (link)] was cultured on Wilkins–Chalgren (WC) Dent agar plates (OXOID, Hampshire, UK) in a microaerophilic atmosphere at 37 °C and 10% CO₂. Cells were infected at a multiplicity of infection (MOI) of 10 (OD₆₀₀ 1 = 2 × 10⁸ bacteria/mL) for 24 h and lysed in sodium dodecyl sulfate (SDS) buffer for protein expression analysis. IFN-γ (10 ng/mL) cells stimulated for 24 h were used as positive control.
Human peripheral blood mononuclear cells were isolated from H. pylori-negative healthy donors, after informed consent, by density gradient centrifugation with Pancoll (PAN-Biotech, Aidenbach, Germany). Cells were cultured with RPMI-1640 containing 10%FCS at 37 °C in a humidified atmosphere (5% CO₂) and infected with the H. pylori G27 strain at MOI 10 for 24 h. The supernatant was collected and co-cultured with gastric cancer cell lines (NUGC4 and NCI-N87) for 24 h. After co-culturing, gastric cancer cells were lysed in SDS buffer for protein expression analysis.

Peripheral blood mononucleated cells (PBMC) were obtained from healthy donor buffy coats (Etablissement Français du sang, Rungis, France) or newly diagnosed CMML patients whose samples were collected with informed consent following the authorization provided by the ethical committee Ile-de-France 1 (DC-2014-2091), separated on Pancoll (Pan-Biotech, Dutscher, Brumath, France). Peripheral blood CD14⁺ monocytes were sorted with magnetic beads and the AutoMacs system (Miltenyi Biotech, Paris, France)^{41 (link)}. Monocytes used for nucleofection experiments were sorted with Monocyte isolation kit II (Miltenyi Biotech). After sorting, monocytes were cultured overnight at 10⁶/mL in RPMI 1640 Glutamax medium (ThermoFisher Scientific) supplemented with 10% heat inactivated fetal bovine serum (FBS, Lonza, Amboise, France), 1% penicillin/streptomycin, and 2 mM L-Glutamine (ThermoFisher Scientific). Cells were then seeded at 0.5 × 10⁶/mL and differentiated into macrophages in the same medium containing recombinant human CSF-1 (100 ng/mL Peprotech, Neuilly-Sur-Seine, France) during 1 to 3 days. Cells were detached using PBS-EDTA and centrifuged at 300×g into dry pellets for further RNA or protein extraction.