Li heparin monovettes

Freshly withdrawn whole blood in Li-heparin Monovettes (Sarstedt) from healthy adult donors that had not received any anti-inflammatory treatment the last 10 days was provided by the Institute of Transfusion Medicine, Jena University Hospital. The blood was incubated for different periods with either BRP-201 or NPs (Ace-DEX, Ace-DEX[BRP-201], PLGA, PLGA[BRP-201]) and stimulated with pathogenic E. coli (O6:K2:H1; 1 × 10⁹ cells per mL blood) for 180 min. The reaction was stopped with ice-cold methanol containing the deuterium-labeled internal standards d8-5S-HETE, d4-LTB₄, d5-LXA₄, d5-RvD2, and d4-PGE₂ (500 pg, each). Samples were kept at − 20 °C for 1 day to allow protein precipitation. After centrifugation (2000×g, 4 °C, 10 min) 8 mL acidified water was added (final pH = 3.5). The samples were subjected to solid phase extraction and analyzed by UPLC–MS–MS as described previously [18 (link)], see above (Sect. Determination of lipid mediator signature profiles in human monocyte-derived macrophages).

Peripheral blood samples were collected with Li-Heparin Monovettes (Sarstedt) and processed within 3 h. Red blood cells were eliminated by osmotic lysis with RBC lysis buffer as described before [2 ]. Leucocytes were then washed, re-suspended in FACS buffer (DPBS + 10% FCS). Unspecific binding of antibodies to cell surface receptors was blocked with Fc Block (1:20, BioLegend) or human TruStain Fc Block (BioLegend) for 20 min at 4 °C. Leucocytes were stained with antibodies specific for CD3 (1:50, BioLegend; UCHT1-PB), CD4 (1:100, BioLegend RPA-T4-PE), CD8 (1:100, BioLegend, SK1-APC), and CD69 (1:50, BioLegend; FN50-FITC) for 25 min at 4 °C in the dark. After washing, the stained cells were fixed for 20 min in 2% PFA, filtered through a 70 µM cell-strainer and characterized by flow cytometry on a LSRII flow cytometer (BD) using a combination of unstained cells, internal negative population controls and single-color staining for color compensation. Therefore, isotype-matched, host-matched monoclonal antibodies were used as negative control: mouse IgG2a, κ (BioLegend; MOPC-173-PE-Cy7), mouse IgG1, κ (BioLegend; MPOK-21-PB), mouse IgG1, κ (BioLegend; MOPC-21-PE), mouse IgG2a, κ (eBioscience; P3.6.2.81-APC). Flow cytometry data was processed with FACS Diva™ software (BD).