Flat bottomed polystyrene plates

An overnight culture of S. aureus MR23 was diluted 500-fold into fresh BHI broth with or without 50 μM of test compound, and 200-μL aliquots were cultured in 96-well flat -bottomed polystyrene plates (Corning) at 37 °C for 24 h with shaking. The absorbance of each culture at 600 nm was measured every 30 min for 24 h using a Bio Microplate Reader HiTS (Scinics Corp., Tokyo, Japan).

E. coli strains were grown in LB medium overnight at 30 °C. The cultures were diluted 1,000-fold in YESCA medium supplemented with the indicated concentrations of EGCG or Myricetin, and 1% DMSO, and 200-μl aliquots were cultured in 96-well flat-bottomed polystyrene plates (Corning, Corning, NY, USA) at 30 °C for 24 h with shaking. The absorbance of each culture at 600 nm was measured every 30 min for 24 h using a Bio Microplate Reader HiTS (Scinics Corp., Tokyo, Japan).

The titration of specific antibodies against rPbHsp60 and rgp43 in the sera from patients with PCM was carried out with indirect ELISA. Sera from NC were used for control. Wells of flat-bottomed polystyrene plates (Corning, USA) were coated with 5 µg of rPbHsp60 or rgp43 diluted in 100 µL of 0.2 M carbonate buffer, pH 9.6, and incubated overnight at 4°C. Then, the wells were blocked with 3% gelatin in TBS-T for 1 h and incubated with 100 µL of serum diluted to 1:250 in a solution of 1% of gelatin in TBS-T, at 37°C. After 2 h, the plates were washed five times and the wells were incubated with 100 µL of alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (Merck Millipore) diluted to 1:2,000, at 37°C. After 1 hour, the wells were washed as described above and the antigen-antibody reaction was detected with a p-nitrophenyl phosphate (pNPP) substrate, according to the manufacturer's instructions (Sigma-Aldrich). The reactions were done in duplicates and the absorbance was determined at a wavelength of 405 nm. The cut-off limit was determined by receiver-operating characteristics (ROC) analysis and was defined as 0.68 absorbance at 405 nm.