Dil oxldl

For characterization of differentiated and polarized macrophages, dead cells were first excluded by live/dead staining (Ghost Dye Violet 510 Viability Dye, Cell Signaling Technology) and single cells were gated by plotting the height against the area of FSC. Cells were single stained with the following antibodies: PE anti-mouse/human CD11b (M1/70; BioLegend), APC anti-human CD163 (GHI/61; BioLegend), FITC anti-human CD197 (G043H7; BioLegend), FITC anti-human CD206 (15–2; BioLegend), PE anti-human CD36 (5–271, BioLegend), FITC anti-mouse CD86 (GL1; BD), Brilliant Violet 421 anti-mouse CD206 (C068C2, BioLegend). The percentage of positive cells and the median of fluorescence intensity were quantified by flow cytometry using FACS Canto II (BD). A minimum of 30,000 cells was assessed. Data were acquired and analyzed using FACSDiva software (BD).
To measure the incorporation Dil-labeled oxidized LDL (oxLDL) by M1- and M2a-like macrophages, cells (1–2 × 10⁵) were cultured for 24 h in HBSS supplemented with 0.3% bovine serum albumin in the presence or absence of 1000 ng/ml NETs. Next, cells were incubated for 6 h with 20 μg/ml Dil-oxLDL (Thermo Scientific) at 37°C, washed twice with PBS and resuspended in 400 μl PBS + 2% FCS+ 1 mM EDTA for immediate FACS analysis.

Cells were plated onto 8-well chamber slides (iBidi, 80826) at a density of 1 × 10⁵cells/well. After the cells became approximately 70 % confluent, media were changed to serum-free DMEM media to starve the cells for 6 hours. Dil-OxLDL (20 μg/mL, Thermo Fisher Scientific, L34358) was added into the media for 2 hours, incubated at 37°C. Cells were then washed 3 times, fixed with 5% Formalin-PBS, and costained with Kim1 antibody.

Murine bone-marrow-derived monocytes were seeded onto 96-well plates and differentiated into macrophages by treating with MCSF and GMCSF for 7 days. After differentiation was complete, macrophages were polarized as described above, or treated with thrombin for up to 48 h. Thereafter, either dil-oxLDL (1:200; Thermofisher, Darmstadt, Germany) or pHrodo Green zymosan bioparticles (1 mg/mL, Thermofisher, Darmstadt, Germany) was added, and phagocytosis was monitored over 24 h using an automated live cell imaging system (IncuCyte; Sartorius, Göttingen Germany).

After polarizations were completed, macrophages were treated with diloxLDL 1:200 (Thermo Fischer Scientific) for 15 min, then fixed with 4% Rotifix, and blocked with 3% horse serum for an hour. At the end of incubation macrophages were incubated with primary antibodies overnight at +4 °C on a shaker, then washed 3 times with 1X PBS. After the washing was completed, macrophages were incubated with secondary antibodies for an hour on a shaker and washed again 3 times with phosphate-buffered saline. Finally, macrophages were incubated with Hoechst (1:1000), and images were taken with a confocal microscope (LSM-780; Zeiss, Jena, Germany) with ZEN Software (Zeiss).

Cells were plated onto 8-well chamber slides (iBidi, 80826) at a density of 1 × 10⁵cells/well. After the cells became approximately 70 % confluent, media were changed to serum-free DMEM media to starve the cells for 6 hours. Dil-OxLDL (20 μg/mL, Thermo Fisher Scientific, L34358) was added into the media for 2 hours, incubated at 37°C. Cells were then washed 3 times, fixed with 5% Formalin-PBS, and costained with Kim1 antibody.

THP-1 cells were trained and differentiated according to the described training protocol. After removal of the differentiation medium, cells were washed in PBS and rested overnight in serum-free RPMI 1640 + Penicillin-Streptomycin. Cells were incubated with 50 µg/mL Dil-oxLDL (Invitrogen) for 4 hours, washed with PBS three times, trypsinized with 0.25% trypsin-0.02% EDTA and stained with eBioscience Fixable Viability Dye eF506 (Thermo Fisher Scientific) in PBS for 15 minutes at 4°C. Cells were analyzed on a LSR Fortessa SOP (BD Biosciences).