Isolation of Polymorphonuclear Leukocytes from Whole Blood

Isolation of cPMNs was performed as previously described [36 (link)]. Venous blood (2 × 10 mL) from controls (n = 13) and periodontitis patients (n = 9) was obtained in lithium heparin tubes (Vacuette® Heparin tubes, Greiner Bio-One, Alphen a/d Rijn, The Netherlands). Blood was diluted 1 : 1 in 1% PBS citrate (pH 7.4). Subsequently, 25 mL of the diluted blood was carefully layered on top of 15 mL Lymphoprep (Axis-shield PoC AS, Oslo, Norway). After centrifugation (800 RCF, 30 min, RT, no brake), the supernatant above the red cell layer was discarded, after which remaining erythrocytes were lysed in cold lysis buffer (NH₄Cl (1.5 M), NaHCO₃ (100 mM), disodium EDTA (1 mM), all Sigma-Aldrich, Merck, Darmstadt, Germany, 10x diluted in sterile Milli-Q (MQ) water). Immediately after erythrocyte lysis, the cPMN pellet was washed twice in cold PBS (Gibco, Thermo Fisher Scientific, Paisley, Scotland, UK) and recovered in a culture medium (phenol red-free, Roswell Park Memorial Institute (RPMI) 1640, Gibco). All samples were handled on the same day without delay.