Fluostar optima fluorescent plate reader

BMDMs were plated at 1.2 × 10⁵ cells per well in 96 well μClear plates (Greiner Bio-One) and treated with 100 U ml⁻¹ IFNγ or 2400 IU ml⁻¹ IFNβ 20 h prior to assay. Fluorescently labeled, IgG-coupled 3-μm silica particles were prepared and used for phagosomal lumenal characterization as previously detailed^{74 (link),75 (link)}. Measurements were performed in microplate format with the use of a FLUOstar Optima fluorescent plate reader (BMG Labtech) or Envision 2104 Multilabel Reader (PerkinElmar) at 37 °C. Immediately prior to the addition of experimental particles, growth medium was removed from cells and replaced with assay medium (DPBS containing 1 mM CaCl₂, 2.7 mM KCl, 0.5 mM MgCl₂, 5 mM glucose, 0.1% calf skin gelatin, and 10 mM HEPES). Particles were added to BMDMs at an MOI of 2-3 particles per cell.

Real-time measurement of intraphagosomal proteolysis as a proxy for phagosomal maturation and function was performed as previously described^{66 (link)}. In brief, 3.0 µm experimental particles bearing human IgG (Sigma Aldrich) and the fluorogenic protease substrate DQ Green BSA (Invitrogen) labelled with the c fluor, AF594SE (Invitrogen) (as described above) were added to cells to achieve a target of 2–3 beads/cell. Phagosomal hydrolytic activity was evaluated by measuring the rate of substrate-liberated fluorescence relative to the calibration fluor. Relative fluorescent units (RFU) as described by the equation RFU = SFRT/CFRT (where SFRT is the substrate fluorescence in real time and CFRT the calibration fluorescence in real time) were plotted against time. Measurements were performed in microplate format using a FLUOstar Optima Fluorescent plate reader (BMG Labtech) at 37 °C. For the evaluation of phagocytic uptake, cells were incubated with 3.0 µm experimental particles bearing human IgG (Sigma Aldrich) and BSA labelled with Oregon Green 488 SE (Thermo Fisher) for 60 min prior image analysis using the IN Cell Analyzer 2000 (GE Lifesciences) automated microscope. Phagocytosis was evaluated by quantifying the number of experimental particles engulfed per cell with the aid of Image J software.

MBCs (96-well plate) were determined based on the levels of oxygen consumption using the FLUOstar OPTIMA fluorescent plate reader (BMG LABTECH Ltd). Each well contained 180 μL of sterile Evan’s media, 10 μL of the specific antibiotic (0.3–2.5 μg/mL amikacin disulphate salt or 0.12–1 μg/mL tobramycin sulphate salt) and 10 μL of an overnight P. aeruginosa culture (0.5 McFarland standard in Evan’s media). Each assay was performed in triplicate. The positive and negative controls had the antibiotics omitted and replaced with either 10 μL of sterile water or 10 μL sodium hypochlorite (reagent grade chlorine 10–15%v/v, Sigma–Aldrich), respectively. The plate was incubated for 72 h at 30 °C.