Phrodo red zymosan bioparticles

Phagocytosis analysis was performed as per supplier’s instructions using pHrodo Red zymosan BioParticles (Life Technologies, Carlsbad, USA) conjugate for phagocytosis. Briefly, isolated WT and Nrf2 KO PMNs (1 × 10⁶/ml) were seeded in duplicate wells, pretreated or not with cytochalasin D (20 μM) (Sigma-Aldrich) at 37 °C with 5% CO₂ for 30 min and then incubated for 90 min in the dark, alone or with increasing concentrations of zymosan bioparticles (5–50 μg/ml). Using flow cytometry (FACS Calibur), phagocytosis was quantified by the increase in particle fluorescence in acidic compartments. Cells were subjected to a one-color analysis (FL-3, PerCP) for the percent of zymosan positive cells.

The phagocytosis assay was performed according to the manufacturer’s instructions using pH-sensitive pHrodo Red Zymosan BioParticles (Life Technologies). The pHrodo Red conjugates do not fluoresce outside the cell at neutral pH but do fluoresce at acidic pH values such as those in phagosomes; this enables an accurate measurement of phagocytosis. Briefly, MGCs were incubated with Zymosan conjugate particles (10 μg/ml) diluted in a live-cell imaging solution (Thermo Fisher Scientific) for 2 h in the dark. Nuclei were counterstained using Hoechst (1:1,000; Life Technologies). After incubation, the cells were thoroughly washed and fixed with 2% paraformaldehyde for 10 min at room temperature. Cell fluorescence was then measured with a microplate reader (SpectraMax M5; Molecular Devices).

For quantification of phagocytosis in vitro, BV2 cells were plated on 96-well cell culture plates (161093, Nunc A/S, Roskilde, Denmark) 7 days after transduction with bicistronic lentiviral particles at a density of 4000 cells/well in 75 µL culture medium. After 16 h, pHrodo Red Zymosan Bioparticles (P35364, Invitrogen, Carlsbad, CA, USA) were added at a final concentration of 5 ng/µL in 25 µL. As a negative control, 2.5 µM phagocytosis inhibitor Cytochalasin D (CytD) was added to two wells per assay. Immediately after addition of pHrodo, fluorescence images were taken every 0.2 h over 1.5 h using a 10× magnification with the IncuCyte S3 Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI, USA) in a humidified incubator at 37 °C in 5% CO₂. IncuCyte S3 software was used to determine phagocytic activity of transduced BV2 cells by quantifying the area of overlapping pHrodo and ZsGreen1 signal and normalizing it to the ZsGreen1 positive area. As negative controls for image analysis, ZsGreen1-negative BV2 cells and BV2 cells without addition of pHrodo Red were included.

M1 polarized macrophages were treated with either solvent or the PPAR-γ antagonist; GW9662 (10 µmol/L, Merck, Darmstadt, Germany), 2 h prior to repolarization to the M2c phenotype using TGF-β1. Thereafter, cells were incubated in RPMI medium supplement with 0.1% BSA (37 °C, 5% CO₂) and containing pHrodo Red Zymosan bioparticles (10 μg/mL, Invitrogen). After 30 min the cells were washed to remove nonphagocytosed material and zymosan uptake was visualized and quantified using an automated live cell imaging system (IncuCyte, Sartorius, Göttingen, Germany).

C57BL/6J BMDM were plated in an eight-chamber slide. Cells were pre-treated with concanamycin A (50 µM) or DMSO carrier for 15 min. Media was then removed, and pHrodo red zymosan bioparticles (Invitrogen) were added at 0.5 mg/ml in BMDM media with concanamycin A or DMSO carrier. After 2 hr, media was removed and cells were washed once with PBS. Cells were then fixed in 4% paraformaldehyde and stained with DAPI (1.25 mu-g/ml). Cells were imaged on a Zeiss Elyra PS.1 microscope with a 20× objective. Images were analyzed using a CellProfiler image analysis pipeline. DAPI-stained nuclei were identified and counted and integrated red pHrodo fluorescence was measured for each image.