1 step turbo tmb elisa substrate

Immuno Maxisorb plates with 96 wells (Nunc; Thermo Fisher Scientific, Rochester, NY, USA) were coated with 10 μg/mL of human collagen I, chicken collagen II, human collagen IV, bovine fibronectin, murine laminin or BSA (Sigma-Aldrich) in PBS at 4 °C overnight and then washed with PBS and blocked with PBS plus 1% BSA for 2 h. Binding of bacteria to laminin coated on plates was studied by using PagN-expressing E. coli. Bacteria grown and induced to make PagN were centrifuged, suspended in PBS to 10⁷ CFU in 100 µL and added to laminin coated wells. Bacteria harboring empty plasmid pRS1 were used as a control. After incubation for 1 h at 37 °C, unbound bacteria were removed by three washing cycles, anti-PagN antiserum (1:500) was added, followed by wash cycles and incubation with goat anti-rabbit HRP-conjugated antibody (1:2000). After three wash cycles, bound antibodies were detected by using the 1-Step Turbo TMB ELISA substrate (Thermo Fisher Scientific) followed by 2 M sulfuric acid and measuring the absorbance at 450 nm. For the binding inhibition assays, double dilutions of heparin or heparan sulfate (400–0.4 µg/mL) were incubated with bacteria for 1 h and the mixtures were added to the laminin-coated wells for further processing as described above.

Transiently transfected cells, used for NFAT reporter assay, were seeded at 50,000 cells per well in poly-l-lysine coated 96-well plates and were grown at 37 °C and 5% CO₂. The next day, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Cells were permeabilized with 0.5% NP-40 (Sigma-Aldrich) for 30 min at room temperature and subsequently blocked for 30 min at room temperature in 1% (v/v) FBS/PBS. Cells were incubated with the rat-anti-HA antibody (1:1000, Clone 3F10, Roche, Basel, Switzerland) for 1 h at room temperature. Subsequently, cells were incubated with Goat anti-Rat IgG-HRP conjugate (1:1000, Pierce, Thermo Scientific) for 1 h at room temperature. Between all incubation steps, cells were washed three times with PBS. 1-Step Turbo TMB-ELISA substrate (Thermo Scientific) was added to the wells and the reaction was stopped with 1 M H₂SO₄. Optical density was measured at 450 nm with a PowerWave plate reader. Data were analyzed using GraphPad Prism version 8.0.

Transfected cells from the NFAT reporter gene assay were seeded in poly-L-lysine-coated transparent 96-well plates. After 24 h, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature (RT), permeabilized with 0.5% NP-40 (Sigma-Aldrich) for 20 min at RT and subsequently blocked using 2% skimmed milk powder in PBS for 1 h at RT. Cells were incubated with anti-HA antibody (rat, clone 3F10, Roche) for 1 h at RT. Subsequently, cells were washed with PBS and then incubated with goat anti-Rat IgG-HRP conjugate (Pierce, Thermo Scientific) for 1 h at RT. After washing with PBS, 1-Step Turbo TMB-ELISA substrate (Thermo Scientific) was added to the wells and the reaction was stopped with 1 M H₂SO₄. Optical density was measured at 450 nm using a PowerWave plate reader (Biotek).