Magnetic separation ls column

NK cells were purified from PBMC of LCL and DCL patients, as well as from healthy donors. Briefly, PBMC were separated by density gradient (Histopaque-1077, Sigma-Aldrich) at 300× g for 20 min at 20°C. Cells were obtained from the interface, washed twice in cold PBS and placed in RPMI-1640 (Gibco), supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 nM HEPES, 100 µg/mL penicillin-streptomycin (Gibco), 17 mM NaHCO₃ and seeded in Petri dishes at 37°C, 5% CO₂ during 18 h for adherence of monocytes. Non-adherent cells were removed, washed in PBS and NK cells were purified with an NK cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, 1×10⁷ total cells was suspended in 40 µL PBS containing 10 µL of cocktail of biotin-conjugated monoclonal antibodies against CD3, CD4, CD14, CD15, CD19, CD36, CD123 and glycophorin A and incubated for 10 min at 4°C. 30 µL PBS and 20 µL anti-biotin microbeads were added for 15 min at 4°C. The cells were washed with PBS, centrifuged at 300×g for 10 min and passed through a magnetic separation LS column (Miltenyi). NK cells were isolated by negative selection. The purity of the enriched NK cells was assessed by flow cytometry using anti-CD56-PE, anti-CD3-FITC (Coulter Immunotech) antibodies, achieving 97% purity. NK cells were washed and plated in 24-well culture-plates.

Neutrophils were isolated from peripheral heparinized blood, obtained from human healthy volunteer donors using Histopaque 1119 and 1077 (Sigma-Aldrich), according to the manufacturer instructions. The granulocyte layer was collected and after washing with cold PBS, erythrocytes were lysed by incubation in lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 10 min on ice, followed by washings with cold PBS and centrifugation at 300 xg for 10 min at 4°C. Neutrophils were highly purified from this preparation by positive selection. Briefly, 5x10⁷ cells were suspended in 50 μl of cold PBS and 50 μl of CD16 micro beads (Miltenyi, Biotec, Bergisch Gladbach, Germany), incubated for 30 min at 4°C, washed with PBS, centrifuged at 300 xg for 10 min and passed through a magnetic separation LS column (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, purified human neutrophils were placed in RPMI-1640 (Gibco) with 2% human serum albumin (CSL Behring) and seeded at 1-2x10⁵ cells on sterile 8-well Cover Chamber Slides (Thermo), and incubated at 37°C under 5% CO₂ atmosphere during 1 or 2 h with the different stimuli to induce NET formation as mentioned below.

Mouse primary pulmonary endothelial cells (mPECs) were isolated, as described (70 (link)). Briefly, lungs were excised, washed, diced, and incubated in digestion buffer (1.5 mg/mL of collagenase A in PBS). Lung lysates were first negatively selected using magnetic CD45-labeled MicroBeads (Miltenyi Biotec; 130-052-301) to remove leucocytes followed by CD31⁺ selection (Miltenyi Biotec; 130-097-418) on magnetic separation LS columns (Miltenyi Biotec; 130-042-401), and they were directly subjected to lysis and mRNA or protein isolation, or to functional analysis using the spheroid angiogenesis assay.