Bactiter glo lysis reagent

The ability of mouse serum or rabbit IgG to inhibit S. aureus binding to immobilized Fg was measured by a previously described assay (57 (link)) with slight modifications. Briefly, 5 × 10⁶ CFU of strain Newman Δspa cells in 60 µl was incubated for 1 h at room temperature with an equal volume of serial threefold dilutions of rabbit IgG (3.33 to 0.013 µg/ml), mouse sera, or phosphate-buffered saline (PBS). The treated S. aureus cells were then added in duplicate to wells of a microtiter plate coated with 2 µg/ml human Fg (Calbiochem) and blocked with 5% skim milk. After incubation for 1 h at room temperature, the plates were washed and incubated for 15 min at 37°C with BacTiter-Glo lysis reagent (Promega), which produces a luminescent signal that is proportional to the amount of ATP present in the sample and corresponds to the number of adherent S. aureus. Luminescence was recorded on a SpectraMax L microplate reader (Molecular Devices, LLC), and the percent binding was calculated by dividing the signal of the antibody-treated S. aureus to that of PBS-treated control bacteria.