Spectramax minimax 300

5 × 10⁵ neutrophils were infected with luciferase-expressing L. (V.) p. strains with different drug-susceptibility phenotypes during 2 h at 34°C at a MOI of 5:1, washed and subsequently incubated for 24 h. Parasite viability after incubation was measured using a luciferase assay system (Promega) according to the manufacturer's instructions. To measure parasite viability in presence or absence of NETs, neutrophils were incubated with or without 10 μg/ml DNase and parasite survival measured by chemi-luminescence as previously described (28 (link)). Chemiluminescence was measured in white, opaque 96-well plates in a plate reader (Molecular Devices, SpectraMax MiniMax 300) at all wavelengths.

3 × 10⁵ Neutrophils were seeded on poly-L-Lysine coated coverslips, primed with 25 ng/mL GM-CSF and exposed for 4 h with either L. (V.) p., PMA (Phorbol-12-myristate-13-acetate, Sigma), PMA + DNase (Sigma) or without a stimulus. Subsequently, cells were fixed with 4% PFA (Paraformaldehyde, Sigma) and stained with rabbit anti-human MPO (Dako) primary antibody and Alexa Fluor-488 goat anti-rabbit secondary antibody (Life Technologies). Coverslips were mounted on glass slides using a DAPI containing mounting medium (Molecular Probes) and analyzed by confocal microscopy (ZEISS LSM 510). To quantify NET formation, NETs were counted using fluorescence microscopy in a blinded manner based on at least in three replicates. NET formation was also assessed through the measurement of dsDNA in the supernatant using the Quant-iT PicoGreen kit (Thermo Fisher) by adapting a technique previously described (42 (link)). 2 × 10⁶ neutrophils were incubated for 4 h in X-vivo medium (Lonza) with either L. (V.) p., PMA or PMA + DNase or without stimulus. After incubation, the cell suspensions were centrifuged, and the supernatants were collected and transferred into black 96-well plates (Perkin Elmer). The picogreen dye was added and fluorescence was measured in using a plate reader (Molecular Devices, SpectraMax MiniMax 300) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Cells were fixed 4 h postinfection with 4% formaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 0.5% bovine serum albumin (BSA) in PBS. The fixed monolayers were serially incubated with monoclonal antibody 2C2 (specific for FMDV nonstructural protein 3A) at 1:1,000 in block buffer, Alexa Fluor 488-conjugated anti-mouse secondary antibody (Invitrogen) at 1:200 dilution in blocking buffer, and the nuclear counterstain TO-PRO-3 (ThermoFisher Scientific). Immunofluorescence images were captured using an imaging plate reader (SpectraMax MiniMax 300; Molecular Devices). Quantitation of the total number of cells based on TO-PRO-3 fluorescence, number of infected cells based on Alexa Fluor 488 fluorescence, and intensity of signal for each cell was automated by image analysis software (SoftMax Pro; Molecular Devices). Data were visualized using Prism 7 (GraphPad).

On day 0, cells at a 70–80% confluence were treated as indicated. At the indicated time points, they were stained with crystal violet, as previously described [65 (link)]. Absorbances were measured at 570 nm on a SpectraMax miniMax 300 imaging cytometer (Molecular Devices, Munich, Germany).