Axioplan 2 fluorescent microscope system

Neutrophils (5x10⁵ cells) were seeded into each well of poly-_L-lysine (Sigma Aldrich)-coated 8-well chamber slides (Nalge Nunc, NY, USA) and incubated for 30 min to allow the cells to attach to the well bottom. Following a medium change, cells were infected with the S. sanguinis strains at an MOI of 10. Exogenous addition of H₂O₂ or 200 nM of PMA served as a positive control. After 3 h of incubation, cells were fixed with PBS containing 4% paraformaldehyde at room temperature for 20 min. Next, cells were blocked overnight with PBS containing 1% bovine serum albumin (Sigma Aldrich) at 4°C and reacted with a goat anti-human elastase polyclonal antibody (1:2000, Santa Cruz Biotechnology, CA, USA) at room temperature for 1 h. After washing with PBS, cells were incubated with Alexa Fluor 594-conjugated anti-goat IgG (1:1000, Molecular Probes, OR, USA) at room temperature for 1 h. Finally, each slide was enclosed with ProLong Gold Antifade Reagent with DAPI (Life Technologies) and observed using a Carl Zeiss Axioplan 2 fluorescent microscope system.

The RBL-2H3 cells were cultured on Cell Desk LF (Sumitomo Bakelite, Tokyo, Japan) in 24 well culture plates and exposed to S. oralis WT, spxB KO (MOI = 200), H₂O₂ (2 mM) or PMA + ionomycin for 3 h, changed to fresh medium containing antibiotics, and cultured for an additional 3 h (total 6 h). The cells were fixed overnight with 10% formaldehyde at 4℃ and stained with Astra Blue dye solution. The Astra Blue dye stains heparin in the granules of mast cells as blue [36 (link)].
The RBL-2H3 cells were cultured as described above, and the viable cells were stained with LysoTracker Red probe (50 nM) and SYBR Green II (1:2000 dilution) in culture medium for 15 min, washed with PBS, and observed using a Carl Zeiss Axioplan 2 fluorescent microscope system (Carl Zeiss, Oberkochen, Germany). LysoTracker Red is an acidotropic red fluorescent probe that accumulates in the acidic lysosomes. SYBR Green is a DNA-binding dyes that stains the nuclei.