Polymorphprep reagent

Neutrophils were obtained from the blood of 15 healthy volunteers (males). All experimental procedures were approved by the Ethics Committee of the Medical University of Bialystok.
Neutrophils were isolated using density-gradient centrifugation with Polymorphprep™ reagent (AXIS-SHIELD PoC AS, Oslo, Norway). Bürker chamber and Türk’s solution were used for counting the cells. Cell purity was assessed by applying the thick drop method, using May-Grünwald and Giemsa dyes. For subsequent isolation, donor cell preparations that exhibited high cell purity with more than 85% neutrophils were used. To obtain a pure cell fraction, the magnetic MACS^® Separator, as well as antibodies and magnetic CD16 MicroBeads (no. catalog no. 130-045-701; Miltenyi Biotec), was used for positive separation. The viability of neutrophils was assessed in preparations formed directly after isolation, as well as in those incubated for 20 h, using trypan blue dye. Sera were obtained from blood samples collected without anti-coagulation agents.

The circulating neutrophils were freshly isolated from eight HFpEF patients and 12 non-HF control individuals. The blood sample (2 mL) was carefully layered over Polymorphprep™ reagent (Axis-Shield, Scotland). After centrifuging at 500 g for 35 min at room temperature, the plasma and mononuclear cells (upper band of cells) were removed, and neutrophils were harvested. After washing with Hepes-buffered saline [0.85% (w/v) NaCl], cell pellet was resuspended in ammonium chloride lysis buffer [0.83% (w/v) NH4Cl, 10 mM Hepes-NaOH, pH 7.4] to remove any residual erythrocyte contamination. Then cells were harvested by centrifugation and stored in TRIzol™ reagent (Thermo Fisher Scientific, USA) for subsequent RNA extraction.