Macsxpress human neutrophil isolation kit

Blood samples were obtained from the pediatric endocrinology and diabetes center at Necker Enfants-Malades hospital, Paris, France in accordance with the approved guidelines. All the experimental protocols were approved by the local ethic committee (CPP - Paris Ile de France, France). Informed consent was obtained from all subjects. Neutrophils were isolated using a MACSxpress human neutrophil isolation kit (Miltenyi Biotec). Red Blood Cell lysis buffer was used to remove residual erythrocytes. The purity of isolated neutrophils was consistently between 98–99% based on CD16 staining. Chemotaxis assay was performed in 24-well micro chemotaxis chamber using 6.5 mm Transwell with 3 μm PVP-free polycarbonate filter membrane (Costar). Neutrophils (2 × 10⁵cells in 200 μl PBS) in upper chamber were allowed to migrate towards 500 μl of conditioned medium produced during 72 h by EndoC-βH2 cultured at pH 7.4 or 6.4. In some experiments, conditioned medium was supplemented with Anti-human IL-8 (1 μg/ml for 10 min; BD554717; BD Bioscience) or with recombinant human-IL-8 (50 ng/ml; BioLegend). After 2 h at 37 °C, migrating cells were recovered with Accutase (Sigma) in the lower chamber and numbered by flow cytometry. Results are expressed as migration index: number of migrating neutrophils in a defined condition divided by number of migrating neutrophils towards pH 7.4 conditioned medium.

Peripheral blood neutrophils from SchS patients (n = 12; canakinumab-treated patients n = 9, untreated patients n = 3; Table S1) and healthy controls (n = 12) were isolated by using a neutrophil isolation kit (MACSxpress® neutrophil isolation kit human, MACS Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany, no. 130-104-434). Neutrophils were assessed for spontaneous NET formation (incubation with RPMI medium with 2% fetal calf serum for 130 min) and for NET formation after stimulation with 20 nM phorbol 12-myristate 13-acetate (PMA) for 80, 100, and 130 min as previously described (11 (link)). Neutrophils were isolated and processed in pairs of one patient and one healthy control at a time.
In addition, NET formation of healthy control neutrophils by SchS sera (10% serum concentration) from symptomatic patients (n = 12), combined with RPMI medium with 2% fetal calf serum as well as sub-threshold (0.05 nM) PMA doses for 130 min, was assessed. Sera from healthy individuals (n = 14) served as controls. Also, NET formation of control neutrophils and neutrophils from SchS patients was assessed after stimulation with IL-1β (10–50 ng/ml), IL-6 (10–100 ng/ml), IL-17 (20–100 ng/ml), or IL-8 (20–100 ng/ml) as well as with a combination of proinflammatory cytokines (IL-1β, IL-6, IL-17, IL-8) and subthreshold PMA.

Eosinophils and neutrophils were collected from whole blood using MACSxpress Neutrophil Isolation Kit, human and Eosinophil Isolation Kit, human, (Miltenyi Biotec) according to the manufacturer’s instructions. Remaining erythrocytes were lysed. Cells were diluted in Complete Medium (CM) consisting of RPMI 1640 (Invivogen, San Diego, CA, USA) with 10% autologous plasma, penicillin 100 U/mL and streptomycin 100 μg/mL (Invitrogen, Carlsband, CA, USA), to a concentration of 1 × 10⁵cells/ml. Migration set up was conducted with transwell plates (3.0 μm PTFE Collagen Coated Membrane, Corning, NY, USA). Cells were added (5 × 10⁵ neutrophils in the bottom well and 2.5 × 10⁵ eosinophils in the insert) and incubated for 3 h at 37 °C in a humidified 5% CO₂ air atmosphere. Inserts were removed and cell suspension in the wells were analysed with flow cytometry using CountBright TM Absolute Counting Beads (Life Technologies, Eugene, OR, USA).

Samples were collected as described previously [14 ]. Briefly, PBMCs were isolated from the peripheral blood by density gradient centrifugation with Ficoll-Paque Plus (GE Healthcare), and erythrocytes were lysed using ammonium chloride potassium buffer, as ammonium chloride potassium buffer has been reported to lyse erythrocytes efficiently without affecting the proportion of immune cells [19 (link)]. After blocking the non-specific binding of antibodies with Fc receptor binding inhibitor (eBioscience), the cells were stained with antibodies shown in Additional file 1The cells were analyzed and sorted using 8-color MoFlo XDP (Beckman Coulter) following the cell surface antigen-based definitions shown in Additional file 2. The results were analyzed using FlowJo version 10.5.0 (TreeStar Software). Neutrophils were isolated from EDTA anti-coagulated blood using MACSxpress Neutrophil Isolation Kit human and MACSxpress Erythrocyte Depletion Kit (Miltenyi Biotec).

Maternal peripheral blood samples were collected 30 min before delivery from the same animals that the fetal membranes were collected. Blood neutrophils were isolated using human MACSxpress neutrophil isolation kit (Miltenyi Biotec, Auburn, CA). Neutrophil purity was >97%, as assessed by flow cytometry and Diff-Quick (Electron Microscopy Sciences, Hatfield, PA) staining of cytospin slides (Supplementary Figure 3). Chorio-decidua neutrophils cells were purified from chorio-decidua cell suspension by FACSAria Cell Sorter (BD Bioscience), using the same gating strategy as for immunophenotyping described previously (10 (link)). FACS-sorted chorio-decidua neutrophil purity was >97% (Supplementary Figure 4), as assessed by Diff-Quick staining. Total RNA was extracted from the purified neutrophils (2–4 × 10⁶) by adding TRIzol (ThermofisherScientific) and subsequently using Direct-Zol RNA MicroPrep kit (ZYMO Research, Irvine, CA) to efficiently extract small quantities of RNA.