Lyse fix buffer 5

For flow cytometry analysis, 50 μl peripheral venous blood was used to obtain 100,000 leukocytes via the inertial microfluidic system (Fig. 1). Leukocytes were incubated for 20 min at RT with the following antibodies to human proteins, with clones noted in parentheses: anti-CD45 PerCP (HI30), anti-CD66b Pacific blue (G10F5), anti-CD16 APC-Cy7 (3G8), anti-CD69 FITC (FN50), pHrodo (PE), anti-CD62L Brilliant Violet 510 (DREG-56), anti-CD42b Alexa Fluor 700 (HIP1) (all from BioLegend) and anti-CD14 APC (61D3) and anti-CD11b PE-Cy7 (ICRF44) (from Thermo Fisher). After staining, the cells were lysed and fixed with 2 ml 1:4 dilution of Lyse/Fix Buffer 5× (BD Phosflow) with distilled water for 15 min at RT. Data were acquired from the BD LSR Fortessa flow cytometer and analysed using FlowJo version 10.1 (Tree Star). PMN (CD45⁺SSC^HiFSC^Hi) subsets were determined by CD16 and CD66b surface expression. Monocyte (CD45⁺SSC^LowFSC^High) subsets were determined by CD14 and CD16 surface expression. The molecules of leukocyte activation were assessed, namely the expression of CD62L, CD11b, CD69 and CD42b.

PMN phagocytic capacity was determined using the pHrodo red E. coli bioparticles conjugate. pHrodo was resuspended with 2 ml PBS (without Ca²⁺ and Mg²⁺) and sonicated for 5 min. After incubation, 2 ml of FBS was added to the pHrodo and incubated for 30 min at 37 °C. After incubation, the particles were pelleted at 1,600g for 5 min and then resuspended with 2 ml PBS (with Ca²⁺ and Mg²⁺) and sonicated for 10 min before use. PMNs (100,000 per FACS tube) from 50 μl peripheral venous blood were isolated using the inertial microfluidic system (see Fig. 1). PMNs were incubated for 20 min at 37 ^oC after exposing cells with 25 μl pHrodo and a total of 4 μl antihuman antibodies to human proteins: anti-CD45 (PerCP), anti-CD66b (Pacific blue), anti-CD16 (APC-Cy7), anti-CD14 (APC), anti-CD69 (FITC), anti-CD11b (PE-Cy7), anti-CD62L (Brilliant Violet 510) and anti-CD42b (Alexa Fluor 700). After staining, the cells were lysed and fixed with 2 ml 1:4 dilution of Lyse/Fix Buffer 5× (BD Phosflow) with dH₂O for 15 min at RT. Data were acquired from the BD LSR Fortessa flow cytometer and analysed using FlowJo version 10.1 (Tree Star).