Summit v4

Binding of monomeric IgG to B2KA cells expressing FcγRI was measured as previously described 1 (link) except that, for the B2 Abs, 80 μg/mL biotin-conjugated goat anti-human λ-chain Abs (Sigma, Poole, UK) were used as the first detection reagent.
Binding to FcγRII and III receptors was measured by pre-complexing the Abs with equimolar amounts of F(ab’)₂ fragments, which recognised the light chain 4 (link): goat F(ab’)₂ anti-human κ (Rockland) for Fog-1 and goat anti-human λ-chain F(ab’)₂ molecules (AbD Serotec or Rockland) for B2 Abs. Human IgA1,κ purified myeloma protein (The Binding Site, Birmingham, UK) or IgA,λ (Jackson ImmunoResearch, Newmarket, UK) were used as negative control test Abs. Complexes were detected using FITC-conjugated F(ab’)₂ fragments of rabbit anti-goat IgG, F(ab’)₂-specific Abs (Jackson ImmunoResearch) or FITC-conjugated donkey anti-goat IgG Abs (AbD Serotec).
Levels of fluorescence were determined using a CyAn ADP flow cytometer and Summit v4.3 software (DakoCytomation, Ely, UK) or on a FACScan flow cytometer and LysisII software (Becton Dickinson, Oxford, UK).

On different days post infection, mice were euthanized and spleens were harvested. Spleen cells were treated with ACK buffer for red blood cell lysis, washed and then stained. Single cell suspensions from spleen were incubated with anti-CD16/CD32 (FcR block) for 5 min and then stained with anti-CD45.1, anti-CD45.2, anti-CD11c, anti-CD3, anti-CD4, anti-CD8, anti-CCR5, anti-TCRβ, anti-T-bet, anti-IL-1R1, anti-IL-18R1, anti-CD62L or anti-CD44 antibodies (see table below) for surface staining for 30 min on ice. Alternatively, 2 × 10⁶ spleen cells were cultured in the presence of monensin (5 μM) and 2 μM of K^b-restricted Tskb20 peptide (Genscript) for 10 hr. After staining of surface markers, cells were fixed with paraformaldehyde 1% for 1 hr and permeabilized with saponin 0.2% for 20 min. At least 20,000 events gated on CD4⁺ T lymphocytes were acquired. Analytical flow cytometry was conducted with a MoFlo (Beckman Coulter/Dako-Cytomation) or FACSCalibur (BD Bioscience) and the data were analyzed with Summit V4.3 software (Dako Colorado, Inc).

To study surface molecules expression, cells were washed with PBS, removed with a cell scraper, and labeled with human FITC-conjugated anti-CD14 (BioLegend, USA) or PE-conjugated anti-CD14 (BD Pharmingen, USA) and PE-conjugated anti-HLA-DR (BD Pharmingen, USA), PE-conjugated anti-CD86 (BD Pharmingen, USA) or FITC-conjugated anti-CD80 (BD Pharmingen, USA), and FITC-conjugated anti-CD40 (BD Pharmingen, USA) or PE-conjugated anti-CD69 (BD Pharmingen, USA) for 30 minutes at 4°C.
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14⁺. Data analyses were performed using Summit v4.3 software (Dako, USA).

The biofilm samples were fixed with 10% sodium azide and prepared for cytometric analysis as described in Patil et al. (2011 (link)). The sample preparation included washing steps to remove the fixative, separation of the cells by vortex and sonication, and staining using a two-step procedure. The first step is 20 min incubation with a solution containing citric acid and Tween20 to facilitate dye penetration and binding. Afterwards, the samples are incubated with the DAPI staining solution (0.68 μM) for at least 60 min.
The flow cytometric measurements were carried out as described before Patil et al. (2011 (link)). A MoFlo cell sorter (DakoCytomation, USA) equipped with two lasers [488 nm and ML-UV (333–365 nm)] was used to analyze FSC, SSC (trigger signal), and DAPI-DNA fluorescence. Fluorescent beads were used to align the instrument: yellow-green fluorescent microspheres (2 μm, FluoSpheres (505/515), Molecular Probes, cat. no. F-8827), blue fluorescent microspheres (1 μm, FluoSpheres (350/440), Molecular Probes, cat. no. F-8815), bright blue Fluoresbrite carboxylate microspheres (0.5 μm (360/407), Polysciences, cat. no. 18339-10). Data acquisition was performed with the Summit v.4.3 software (DakoCytomation, USA).

Monocytes were isolated from PBMCs using CD14 MicroBeads (Miltenyi Biotec) and activated with LPS or LRZ. Cytofluorimetry was performed as indicated^{57 (link)}. Cells were stained with anti-human LAMP2A Ab (Abcam) followed by the anti-rabbit FITC secondary Ab (Bethyl Laboratories, Inc). Samples were acquired using a CyAn flow cytometer (Beckman Coulter) and analyzed by the Summit V4.3 software (DAKO)^{32 (link)}.

After 24 hours of incubation in the absence or presence of 10⁻⁷ M of ouabain, cells were incubated with 1 mg/mL FITC-conjugated dextran (Sigma, USA) for 1 hour at 37°C or 4°C (for control endocytic activity). Cells were then washed with ice-cold PBS to remove free dextran particles and fluorescence was analyzed by flow cytometry. Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate. Data analyses were performed using Summit v4.3 software (Dako, USA).