Samples were mixed with respective reaction buffers and read at fluorescence at Ex/ Em=535/590 nm in a microplate reader to measure products concentrations.
The suspension containing HCP was applied onto a molecular exclusion chromatography column (Sephadex G-100; Sigma- Aldrich) and the pili were eluted in 0.5 ml fractions with 100 mM sodium phosphate buffer (pH 7.4), and read at OD280 nm.
Plates were incubated for 1 h, washed with buffer, incubated with 1 5000 secondary anti-rabbit antibody conjugated with horseradish peroxidase, washed, mixed with detection buffer TMB (Alpha Diagnostic), and read at OD 450nm in a BioTek plate reader.
The level of the superoxide anion radical (O2−) was measured by Nitro Blue Tetrazolium (NBT) reaction in TRIS-buffer with coronary venous effluent and read at 530 nm.
After washing, the plates were stained with pNPP (1 mg/ml p-nitrophenyl phosphate in coating buffer, 0.1 N Na2CO3/NaHCO3; pH 9.6) and read at 405 nm (reference wavelength 620 nm).
After washing 5×, plates were developed using 100 µl/well o-phenylenediamine in phosphate-citrate buffer, stopped with 100 µl/well 1 N H2SO4 and read at 490/620 nm.
Wells were washed four times in washing buffer, and color was developed using the TMB substrate (Cell Signaling) and read at 450 nM wavelength.
After washing 5×, the plates were developed with 100 µl/well o-phenylenediamine in phosphate-citrate buffer (Sigma-Aldrich), stopped with 100 µl/well 1 N H2SO4 (VWR, West Chester PA, USA) and read at 490/620 nm.
After a final wash, para-nitrophenyl phosphate (pNPP) substrate buffer (Sigma-Aldrich) was applied, and the plates were incubated for 30 minutes at RT and read at 405 nm on a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA).
The medium was removed from cells, and dissolved in DMSO (DMSO 0.1% in phosphate saline buffer) and read in an ELISA micro plate reader at 570 nm; 48 h after treatment, viability was accessed by MTT assay.
