Phagolysosome Formation in Response to Resolvin D1 and D2

PMN phagolysosome formation response to RvD1 and RvD2 in disease and health was assessed by using pHrodo red E. coli bioparticles conjugate. The preparation of pHrodo red E. coli bioparticles conjugate is described in previous work (7 (link)). Using the inertial microfluidic separation, 100,00 leukocytes were isolated from 50 μL of peripheral blood. Leukocytes were first incubated for 5 minutes at 37°C. Following incubation, cells were exposed to RvD1 (1, 10, or 100 nM), RvD2 (1, 10, or 100 nM), or vehicle (<0.01% v/v EtOH) for 15 minutes at 37°C. Leukocytes were then incubated for 15 minutes at 37°C after exposing cells with 25 μL of pHrodo. Cells were washed with 1 mL of PBS (without Ca²⁺ and Mg²⁺) and resuspended with 50 μL of PBS (without Ca²⁺ and Mg²⁺). A total of 2 μL of anti-human antibodies to human proteins were used to stain leukocytes, with clones noted in parentheses: anti–CD45 PerCP (HI30), anti–CD66b Pacific Blue (G10F5), anti–CD16 APC-Cy7 (3G8), and anti–CD14 PE-Cy7 (63D3) (all from BioLegend). After staining, the cells were lysed and fixed with 2 mL of 1:4 dilution of Lyse/fix Buffer 5X (BD Phosflow) with distilled water for 15 minutes at RT. Data were acquired on a BD LSR Fortessa flow cytometer and were analyzed using FlowJo software version 10.1 (Tree Star Inc.).