Cd14 pe clone m5e2

20 mL of the whole blood on lithium heparin anticoagulant was collected from HIV-infected individuals and HC. Neutrophils were purified by negative selection by microbeads, which allowed the removal of DCs, B cells, monocytes, macrophages, activated T cells, and activated NK cells (MACSxpress Whole Blood Neutrophil Isolation Kit; cat. 130-104-434). Residual erythrocytes were lysed with the use of 2mL ammonium chloride Lysing Reagent (BD cat. 555899) for 5 minutes. The final purity of PMN population was assessed by flow cytometry using CD14-PE (clone M5E2), CD15-FITC (MMA), and CD16-PECy7 (3G8, all from BD Pharmingen) mAbs. Flow cytometric analysis of isolated populations of cells showed that the percentage of CD15^highCD16⁺CD14^- neutrophils was >98%. The level of contaminating CD14⁺CD15⁺ monocytes was about 0.4% and CD15⁺CD16^- eosinophils was <0.1% after isolation (Figure S1A in Supplementary Material). 2x10⁶ neutrophils were incubated without stimulation, in the presence of 100ng/mL ultrapure LPS from E. coli (serotype R515, Alexis Biochemicals) in RPMI 1640 for 6 h (5%CO₂, 37°C, humid atmosphere). For DNA isolation, samples of isolated neutrophils were frozen and kept at -150°C.

We collected 20 mL of whole blood on lithium heparin anticoagulant from sepsis, NMOSD, and periodontitis patients as well as healthy controls (HC). Neutrophils were purified by negative selection by microbeads, which allowed the removal of DCs, B cells, monocytes, macrophages, activated T cells, and activated NK cells (MACSxpress Whole Blood Neutrophil Isolation Kit, Miltenyi Biotec GmbH, Germany). Residual erythrocytes were lysed with the use of 2 mL ammonium chloride Lysing Reagent (BD Biosciences) for 5 min. The final purity of PMN population was assessed by flow cytometry using CD14-PE (clone M5E2), CD15-FITC (MMA), and CD16-PECy7 (3G8, all from BD Pharmingen, San Diego, CA, USA) mAbs. Flow cytometric analysis of the isolated population of cells showed that the percentage of CD15^highCD16⁺CD14^- neutrophils was >98%. The level of contaminating CD14⁺CD15⁺ monocytes was about 0.4% and CD15⁺CD16^- eosinophils was <0.1% after isolation (Figure S1A in Supplementary Material).

The immunophenotype of primary BM CD138⁺ cells, HMCLs and microenvironment cells was analyzed with the following mAbs:
CD38-APC (clone HIT2, code n. 560677, BD)
CD31-FITC (clone WM59, code n. 555445, BD)
CD39-APC (clone eBioA1, code n. 17-0399-41, eBioscence; San Diego, CA)
CD73-APC (clone CB73), produced in the Lab of one of the authors (FM) and FITC-conjugated by AcZon (Bologna, Italy)
CD203a-FITC (clone 3E8, kindly provided by J. Goding)
CD14-PE (clone M5E2, code n. 555398, BD).