Facd diva

Isolated PMNL (2 × 10⁶ cells/ml) were transferred in 250 μl RPMI 1640 with supplements as described above including 20% serum from TP. Control samples were incubated in medium containing supplements without sera, and as an additional control IL-8 was applied to the medium. The samples were incubated at 37 °C and 5% CO₂ for 2 hours. Subsequently, the samples were centrifuged at 2100 x g for 15 min, the supernatant was removed and the cells were transferred into polystyrene FACS tubes. Then, the samples were incubated with mouse anti-human CD62L APC (clone: DREG-56), mouse anti-human CD31 (clone: WM59) and mouse anti-human CD11b/Mac-1 PE (clone: ICRF44, all BD Pharmingen) antibodies for 30 min. After subsequent washing procedure with 4 ml PBS supplemented with 0.5% bovine serum albumin (FACS buffer) at 400 x g for 5 min, the supernatants were removed and the cells were resuspended in 200 μl FACS buffer. Flow cytometric analyses of cell surface markers on a minimum of 20.000 cells was performed using BD FACS Canto 2™ and FACD DIVA™ software (BD). The neutrophils were gated by the corresponding forward and side scatter scan (FSC and SSC) as shown in Fig. 2 a. Mean fluorescence units (MFU) were detected.

Isolated neutrophils (2 × 10⁶ cells/ml) were cultured as described above with/without 20% serum from trauma patients in culture media. Control samples were incubated in medium without serum from patients. The samples were incubated at 37 °C and 5% CO₂ for 2 h later centrifuged at 2100 g for 15 min, resuspended in culture medium (1 ml) with supplements and 100 μl of the cell suspension were transferred into polystyrene FACS tubes. Twenty μl CM-H2DCFDA (CM-H₂DCFDA, General Oxidative Stress Indicator Kit, Invitrogen, Darmstadt, Germany) were added to each sample as suggested by the manufacturer. Subsequently, the samples were incubated for 30 min at 37 °C and 5% CO₂. Thereafter, 400 μl of cell culture medium with supplements (as described above) were added to each sample. After 60 min at at 37 °C and 5% CO₂, the cells were washed with 4 ml FACS buffer and centrifuged at 400 g for 5 min. The supernatants were removed, the cells diluted in 200 μl FACS buffer and subjected to flow cytometry using BD FACS Canto 2™ and FACD DIVA™ software (BD). The neutrophils were gated by the corresponding forward- and side scatter scan. From each sample a minimum of 20.000 cells was measured. The amount of positive cells for oxidative stress was calculated relative to the whole neutrophil population of unstained cells as percentage in representative figures.

100,000 cells per polystyrene FACS tube (BD Pharmingen™) were treated according to the Monocyte treatment section with slight change. After 1 h of treatment with sera, Brefeldin A (Invitrogen) was added to each tube to 1x concentration and monocytes were incubated for further 2 h at 37°C and 5% CO₂. Subsequently, monocytes were incubated with Phycoerythrin-conjugated anti-human CD14 antibody (2 μL; Clone M5E2; BioLegend) and fixable yellow dead cell stain (2 μL; Invitrogen, Carlsbad, California, US). After 30 min at RT, cells were washed with FACS buffer and centrifuged at 400 g for 5 min. Supernatant was removed and monocytes were fixed with Fix and Perm Medium A at room temperature for 15 min. After further washing step, cells were permeabilized with Fix and Perm Medium B (both Invitrogen, Carlsbad, California, US) and incubated with PerCP/Cyanine5.5-conjugated anti-human TGF-β1 antibody (2 μL; Clone TW4-2F8; BioLegend) at room temperature for 30 min. Following the washing step, monocytes were resuspended in 50 μL FACS buffer and analyzed using BD FACS Canto 2™ and FACD DIVA™ software (BD). Monocytes were gated by the corresponding forward and side scatter scan and as shown in Figures 5A,B. The percentage of TGF-β1 expression of viable CD14⁺ monocytes was analyzed.