Poly l lysin

THP-1 cells were seeded on glass coverslips in bottom chamber 4-well slides (ibidi) and incubated with poly-L-lysin (Sigma Aldrich, Deisenhofen, Germany) for 1 h. Then, the cells were fixed with 4% paraformaldehyde (PFA) (ThermoFisher Scientific) for 15 min, permeabilized with 0.1% Triton X-100 in PBS/BSA 1% blocking buffer (Cell Signaling Technology, Danvers, MA, USA) for 30 min at RT, and incubated overnight at 4 °C with primary antibodies against CD74 (AF7478) (R&D, Minneapolis, MN, USA) and TIMP-1 (ab1827) (Abcam, Cambridge, Great Britain). Non-specific isotype antibodies were used as negative controls. Species-specific Star488/Star635 secondary fluorescence antibodies (Abberior, Goettingen, Germany) were applied for 2 h at RT. The slides were embedded in Prolong^® Diamond antifade mountant (ThermoFisher Scientific) in the presence of 4′,6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei. Digital images were acquired using a Leica DMi8 fluorescence microscope equipped with a digital camera (Leica Microsystems).

IgG1, IgG2a, IgG2b and IgG3 antibody secretion directed against dsDNA was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 384-well microtiter plates (Greiner Bio On, Frickenhausen, Germany) were pre-coated with 20 µg/mL Poly-L-Lysin (Sigma-Aldrich, Chemie, Taufkirchen, Germany) for 1 h at 37 °C followed by coating with 20 µg/mL calf thymus DNA (Sigma-Aldrich) at 4 °C o.n. Plates were blocked with 2% fetal calf serum (FCS) in PBS for 2 h at RT. Samples were diluted in 2% FCS in PBS and incubated for 2 h at RT. Bound α-dsDNA immunoglobulins were detected with HRP-conjugated secondary antibodies specific for mouse IgG1, IgG2a, IgG2b or IgG3 (Southern Biotech, Birmingham, Alabama, USA), followed by development with TMB substrate (Thermo Fisher Scientific, Freiburg im Breisgau, Germany) according to the manufacturer’s protocol. The absorbance at 450 nm was measured using the Spark® 10 M multimode microplate reader (Tecan, Crailsheim, Germany). To determine autoantibody titers, expressed as arbitrary units (A.U.), reference sera were used to create a standard curve.

Primary antibody AQP1 was obtained from Abcam Company (UK). Anti-rabbit HRP/DAB Detection kit was obtained from Bethyle Company (USA). IL6 ELISA Kit (rat IL-6 platinum ELISA) was obtained from Bender Medsystems Company (Austria). Phosphate buffer saline was obtained from Gibco-Invitrogen Company (UK). Blood serum bovine, hematoxylin, poly-l-lysin and eosin were obtained from Sigma Company (USA). Xylene, Tween, Triton, formalin, paraffin, ethanol, methanol and chloroform were obtained from Merck Company (Germany). MTX was obtained from Kocak Farma (Turkey).

MicroNB-shControl and MicroNB-shPHOX2B cells were seeded onto a 96-well ImageLock tissue culture plate (Essen BioScience, Ann Arbor, MI, USA) which was pre-coated with 0.015% Poly-L-lysin (Sigma-Aldrich Corp., St Louis, MO, USA). Cells were allowed to attach over a 24hr period, then wounds were made with the 96-well WoundMaker (Essen BioScience). The culture plate was washed once with PBSx1 to remove detached cells, and a fresh growth medium was added. Images of the wounds were automatically acquired within the CO₂ incubator by IncuCyte zoom (Essen BioScience). Typical photomicrographs were taken at 2hr intervals for a 72hr period. The data were analyzed with respect to wound confluence and calculated using the IncuCyte software (Essen BioScience).

For live cell imaging, 3 clones of each Nr0b1^KO/Y::Zscan4c-mCherry and Nr0b1^fl-/Y::Zscan4c-mCherry ES cells were seeded 1000cells per well on a ibidi 8well chamber (Nippon Genetics) coated with poly-L-lysin (100 μg/ml; Sigma-Aldrich) and then with Ecadherin-Fc (5 μg/ml; R&D Systems). Cells were monitored in a humid atmosphere with 5% CO₂ at 37°C under an inverted microscope (IX81, Olympus) equipped with a confocal spinning disk (CSU-X1, Yokogawa), a CCD camera (iXon, Andor) and MetaMorph imaging software (Molecular Devices). Time lapse images were taken every 1 hour with a 10× objective lens.

Living specimens were cold-anesthetized for several minutes and prefixed for 10 min at room temperature in 4% paraformaldehyde (PFA) (Sigma Aldrich, #P6148, St. Louis, MO) in sodium hydrogen phosphate buffer (PBS, 0.1 M, pH 7.4; chemicals obtained from Carl Roth, Karlsruhe, Germany). Subsequently, specimens were decapitated and the VNC was dissected with fine forceps and fixed in 4% PFA dissolved in PBS over night at 4 °C.
Four specimens of each species were treated as whole mounts, two additional specimens of each species were further processed for vibratome sectioning. For the latter, preparations were washed in PBS, overlaid with Poly-L-Lysin (Sigma Aldrich, #P8920) for several minutes and embedded in 4% agarose (Sigma Aldrich, #A9414) dissolved in PBS at approximately 40 °C. After cooling to room temperature, the trimmed agarose-blocks were sectioned horizontally (80 μm) using a Microm HM 650 V vibratome (Thermo Scientific, Eugene, OR).