White 96 well plate

All luciferase assays were performed with the Nano-Glo Luciferase Assay kit (Promega). For infected ALI cultures, transwells were incubated in 100 μl Nano-Glo Luciferase buffer (top compartment only) at 37°C for 15 mins. Cells were then scraped from the transwell membrane with a blunt pipette tip and transferred to a single well of a white 96-well plate (Greiner Bio-One). 100 μl of a 25:1 Nano-Glo Luciferase buffer to Nano-Glo Luciferase substrate mix was added to each well, and the plate was incubated for 3 min at room temperature. Luminescence values were read on a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek). For mouse fecal pellets in 1.7 ml microcentrifuge tubes, pellets were ground with a pestle, then 3-mm glass beads (Fisher Scientific) and 1 ml fecal lysis buffer (50 mM Tris pH7.6, 2 mM DTT, 2 mM EDTA pH 8.0, 10% glycerol, 1% Triton X-100 prepared in water) (Pawlowic et al., 2017 ) were added to the tube. Tubes were incubated at 4°C for 30 mins, vortexed for 1 min, then spun at 16,000 x g for 1 min to pellet debris. 100 μl supernatant was added to two wells of a white 96-well plate, then substrate addition and luminescence reading was performed as above.

For luminescence-based ATP measurements, MEF were seeded at a density of 1.9 × 10³ cells/well in a white 96-well plate (Greiner, Kremsmünster, Austria). ATP levels were analyzed by luminescence detection (FluoStar; BMG Labtech, Ortenberg, Germany) according to the manufacturer’s protocol using the ViaLight plus kit (Lonza, Basel, Switzerland) after 48 h and 72 h of OH-TAM incubation. Data are representative for three independent experiments (n=7 per treatment condition).

Transient transfection was performed using TurboFect transfection reagent (Thermo scientific, Dreieich, Germany) according to manufacturer’s instructions. The ratio of DNA to TurboFect employed was 2 μg DNA per 4 μl transfection reagent. For transfection, cells were first seeded in 96-well plates at a density of 10,000 cells per well in 200 μl of medium. After 24 hours, old medium was removed and fresh medium containing the DNA/Polymer complexes was added (100 μl with an equivalent of 25 ng DNA). Three hours later, medium was exchanged and the cells received fresh medium with the supplements to be tested. Gaussia luciferase activity was determined from the supernatant 24 hours after the start of the transfection as described before [26 (link)]. Briefly, cell supernatant (5 μl) was transferred into a well of a white 96-well plate (Greiner bio-one, Frickenhausen, Germany). After an incubation of 20 minutes at room temperature, Gaussia luciferase activity was determined using a Mithras LB 940 Multimode reader (Berthold Technologies, Bad Wildbad, Germany) by injecting 50 μl of luciferase assay reagent (20 mM MOPS (3-(N-morpholino)propanesulfonic acid); 0.75 M KBr; 5 mM MgCl₂; 5 mM CaCl₂; 1 mM EDTA (ethylenediaminetetraacetic acid); 10 μM Coelenterazine; pH 7.8) to the cell supernatant, followed by a 1.6 second delay until luminescence was determined within a 0.5 s integral.

For measurement of ROS production, 30,000 BMDCs were seeded per well of a white 96 well plate (Greiner) in a volume of 50 µl in RPMI medium without FCS and phenol red (Gibco). Cells were allowed to settle for 1 h. To induce ROS production, cells were treated with 50 µg/ml depleted Zymosan, 50 µg/ml Zymosan, 200 µg/ml Curdlan, MOI 10 HKCA or 500 ng/ml LPS, which were added to the cell suspension in a total volume of 50 µl as a twofold concentrated solution. Additionally, these solutions were supplemented with 100 µM L-012 (Tocris) serving as luminescent probe for ROS detection. In blocking experiments, cells were incubated with 10 µg/ml anti-Dectin-1 (2A11, Biorad) for 30 min prior to ligand application. Luminescent signals were detected either employing Biotek Synergy, Infinite M Plex (Tecan), Victor^{3 (link)} 1420 (Perkin Elmer) or CLARIOstar Plus (BMG labtech) multi-mode readers every 2.5 min for a total of 90 min. ROS production was quantified as Area under the curve (AUC), which was calculated using GraphPad Prism software.

To measure the induction of apoptosis in infected cells, activity of the apoptotic markers caspase-3 and −7 was measured at different time points p.i. (2 h, 7 h, 12 h, 24 h) using a commercial Caspase-Glo 3/7 assay kit (Promega, Madison, Wisconsin, United States) following the manufacturer’s instructions. Briefly, HUVEC were collected at different time points and 5000 cells were added to a white 96-well plate (Greiner, Germany) in duplicate. As a positive control, cells were treated with staurosporine (0.25 µM; Biaffin GmbH & Co KG, Germany) for 3 h. The caspase 3/7 substrates were added to each well and the plate was shaken at 300 rpm for 2 min followed by incubation in the dark for 30 min. Luminescence was quantified using a Synergy 2 multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT), and the results were expressed as n-fold induction relative to the uninfected control cells.

To test the polymer’s cell viability, a CellTiter-Glo^® Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA) was performed, according to the manufacturer’s protocol. The assay was performed using the L929 mouse fibroblast cell line. Cells were seeded in a white 96-well plate (Greiner Bio-One) at 8500 cells per well. After 24 h of incubation, a dilution series from 3.9 µg mL⁻¹ to 500 µg mL⁻¹ of the polymer was prepared in RPMI 1640 (+10% FCS), and 100 µL of each concentration was added to the wells. Subsequently, the cells were incubated for further 24 h. After one day of incubation, 100 µL of the CellTiter-Glo^® reagent was added, following 2 min of shaking at 450 rpm and 10 min of incubation without shaking. Luminescence was determined with the Tecan Spark 20M (Tecan Group AG). A thiomersal 0.02% solution (Caelo, Hilden, Germany) served as a positive control, and untreated cells were set as 100% control. RPMI 1640 supplemented with 10% FCS served as the blank value and was subtracted from all the measured values. The final cell viability was calculated by setting the measured values in correlation to the untreated control. Cell viability values below 70% were defined as toxic, according to DIN EN ISO 10993-5 [36 ]. This assay was repeated twice with eight technical replicates, and data are shown as percentages (mean ± SEM).