Neutrophil isolation and labeling

Experimental protocols were approved by the Research and Ethics committee at Arabian Gulf University. All methods were carried out in accordance with the committee's relevant guidelines and regulations. Participants provided written informed consent prior to enrolment in the study and for the publication of the data. Isolation of neutrophils was performed using Polymorphprep™ density gradient solution (Progen Biotechnik GmbH) to isolate polymorphonuclear granulocytes from whole blood (44). Briefly, 30 ml whole blood from a healthy human volunteer were collected in EDTA and 5 ml of whole blood were layered over 5 ml Polymorphprep™ solution and centrifuged at 450 × g for 30 min at 18–22°C. The plasma and upper leukocyte phase containing peripheral blood mononuclear cells were removed and the lower leukocyte layer containing neutrophils was recovered. Cells were washed in PBS (without Ca²⁺ and Mg²⁺) and centrifuged at 450 × g for 10 min at 18–22°C. After red blood cell lysis, cells were washed in 1X PBS (without Ca²⁺ and Mg²⁺), then centrifuged at 250 × g for 5 min at 18–22°C. After a final wash in PBS (without Ca²⁺ and Mg²⁺), cells were resuspended at 2×10⁶ cells/ml in RPMI-1640 (Sigma-Aldrich; Merck KGaA).

2′,7′-Bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, acetoxymethyl (BCECF-AM; Invitrogen; Thermo Fisher Scientific, Inc.) was used for neutrophil labeling. Briefly, BCECF-AM was added to the cells at a final concentration of 1 µM and incubated for 30 min at 37°C. Cells were washed twice in HBSS/BSA and resuspended in serum-free RPMI-1640 medium (Sigma-Aldrich; Merck KGaA) to achieve a final concentration of 1×10⁶ cells/ml.