Varioskan flash reader

cf-DNA was measured to evaluate NET formation [28 (link)]. For quantification of cf-DNA, 200 μl of freshly extracted synovial fluid was used. Samples were incubated with 0.5 U/ml per well of micrococcal nuclease (New England Biolabs, USA) for 30 min at 37 °C. Then samples were centrifuged at 500×g for 5 min at RT. Afterwards, 100 μl of supernatant was transferred to a new well and 100 μl of Quant-iT™ PicoGreen™ dye (Thermo Fisher Scientific, MA, USA) diluted in TE buffer (10 mM Tris, 1 mM EDTA) was added. Analysis was performed using an automated Varioskan Flash Reader (Thermo Fisher Scientific, MA, USA) at 484 nm excitation and 520 nm emission, as described elsewhere [29 (link)].

Quantification of extracellular free DNA was measured by spectrofluorometry. PMNs (1 × 10⁶) resuspended in Hanks' Balanced Salt Solution (HBSS, 136 mM NaCl, 5.3 mM KCl, 5.5 mM glucose, 1 mM MgCl₂, 10 mM HEPES, 1.2 mM CaCl₂, pH 7.4) were incubated in the presence of tamoxifen (TX, 10 μM, Sigma-Aldrich) or DMSO (0.1%, drug vehicle) for 30 min at 37°C with 5% CO₂. Then, to stimulate NETs formation (positive control), cells were incubated with phorbol-12-myristate-13-acetate (PMA; 200 nM, Merck Millipore) for 1 h at 37°C with 5% CO₂. Next, micrococcal nuclease (5U/well, New England Biolabs, Ipswich, MA, USA) was added to each sample, and incubated for 15 min at 37°C with 5% CO₂. Cells were centrifugated at 300 × g for 5 min, and 100 μL of the supernatants were collected and transferred to a 96-well plate. PicoGreen^TM (1:200, Invitrogen, Carlsbad, CA, USA) was added to each well, followed by a 5 min incubation at RT. DNA content and NETs formation was analyzed using an automated Varioskan Flash Reader (Thermo Fisher Scientific, MA, USA) at 485 nm excitation and 530 nm emission.

In the C16-HSL bioassay, C. violaceum VIR24/pPROBE-PvioA-gfp (Toyofuku et al., 2017b (link)) and P. denitrificans Pd1222ΔpdnIΔpxm/pPLlas were used as C16-HSL reporter strains. In Pd1222 ΔpdnI/pPLlas, gfp(ASV) expression is induced in response to exogenous C12- to C18-HSL (Toyofuku et al., 2017b (link); Morinaga et al., 2018 (link)). Pxm is involved in extracellular matrix production in P. denitrificans Pd1222 (K. Morinaga, unpublished). Ten microliters of the filtered supernatants of the P. denitrificans Pd1222 culture was added to 1‍ ‍mL of medium in each well of a 24-well flat-bottomed plate. Overnight cultures of the reporter strains were inoculated at an OD₆₀₀ of 0.01. The supernatant and reporter strains were incubated at 400‍ ‍rpm at 30°C for 12 h. Synthetic C16-HSL (Cayman Chemical) was added at final concentrations of 0–10‍ ‍μM. After the incubation, the cell culture was centrifuged at 16,000×g at 4°C for 1‍ ‍min. Cells were then washed, dissolved in PBS, and placed in a 96-well black plate (Grainer Bio-one). QS promoter activity was calculated by normalizing RFU (Ex/Em=475‍ ‍nm/515‍ ‍nm) with the OD₆₀₀ value, measured using a Varioskan Flash Reader (Thermo Scientific).

Activity of lectin(s)-MASP-2 complexes was determined as previously described (43 (link)) with modification (44 (link)). LPS from various bacteria were used for coating of microtiter plates (Maxisorp U96, Nunc). The products of C4 activation were detected with rabbit anti-hC4c and HRP-conjugated anti-rabbit Ig (Dako). To test MBL-MASP-1 complex activity, VPR-AMC (Val-Pro-Arg-aminomethylcoumarin) peptide (Bachem, Switzerland), as the substrate for MASP-1 was used as previously described (45 (link)) and the fluorescence was read using a Varioskan Flash reader (Thermo Scientific, USA).

Caspase-3 activity was quantified using the Caspase-3/CPP32 Fluorometric Assay Kit (BioVision, Milpitas, CA, USA) as per the manufacturer’s instructions. OVCAR3CIS (3.0 × 10⁴ cells/mL) or SKOV3ip1CIS (3.5 × 10⁴ cells/mL) were seeded into 10 cm Petri dishes and transfected with NC-siRNA or CASC10-siRNA(2). After 24 h, the media was replaced by regular media, and seventy-two hours after transfection, cells were collected, pellets were lysed, and total protein concentration was determined. Equal amounts of protein were mixed with 2X Reaction Buffer and 1mM DEVD-AFC substrate in a 96-well plate and incubated at 37 °C for 2.5 h. Fluorescence intensity at 400 nm excitation and 505 nm emission was measured using the Varioskan Flash reader from Thermo.

Serum ficolin-1 and ficolin-3 concentrations were determined by TRIFMA [as described by Wittenborn et al. (7 (link))] and ELISA [according to Michalski et al. (47 (link))], respectively. Ficolin-2 levels were measured by TRIFMA. Briefly, microtiter plates (Optiplate-384HB, Perkin Elmer, Waltham, MA, USA) were coated with antificolin-2 (ABS 005-16, BioPorto Diagnostics, Copenhagen, Denmark, 1 μg/ml). Plates were blocked with 0.1% BSA and then incubated with sera to be tested, pre-diluted 1:10. Biotinylated mAb (clone GN4, 1:100, Hycult Biotech, Uden, The Netherlands) and Eu³⁺-labeled streptavidin (Perkin Elmer) were used for detection. After incubation with the enhancement solution (Perkin Elmer), fluorescence values were measured using Varioskan Flash reader (ThermoFisher Scientific, Waltham, MA, USA). Serum from a healthy volunteer (ficolin-2 concentration: 3,500 ng/ml) was used as a standard.
“Low” and “high” values were arbitrarily based on 10 and 95th percentiles, respectively, determined for the control group. Consequently, concentrations <620 ng/ml (ficolin-1), <1,670 ng/ml (ficolin-2), and <12.9 μg/ml (ficolin-3) were considered “low,” while concentrations >2,900 ng/ml, >6,350 ng/ml, and >34.9 μg/ml, respectively, were considered “high.”