Alexa fluor 546 conjugated donkey anti mouse igg h4l

S. aurantiacum and P. minutispora cells, fixed in 4% paraformaldehyde cacodylate buffer (0.1 M, pH 7.2) for 1 h at room temperature, were blocked using PBS-1%BSA for 1 h at 37 °C. Then, either anti-GlcCer Mab or an isotype-matched control (50 mg/mL in PBS-1%BSA) was used to check GlcCer exposure on the fungal surface. After washing, cells were treated with Alexa Fluor 546-conjugated donkey anti-mouse IgG (h4l) (Invitrogen Molecular Probes, Carlsbad, CA, USA) at 1:400 dilution in PBS-1% BSA for 1 h at 37 °C. Cells were washed three times and suspended in 0.01 M N-propyl gallate diluted in PBS: glycerol (1:1, v/v). The suspension was applied to a microscope slide, and cells were visualized using an Olympus AX70 fluorescence microscope (Olympus America Inc., Center Valley, PA, USA) using a 620 nm filter and a 100× magnification lens.

MTT [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] and paraformaldehyde were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Peritoneal macrophages were obtained from male BALB/c mice (4–8 weeks) and maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS). The RAW and A549 cells were maintained in DMEM. In all experiments, the cell counts and viability were determined by trypan blue vital dye exclusion using a hemocytometer. This method yielded conidia that had an initial viability of >95%, as confirmed by plating. Peptidorhamnomannan (PRM) was produced as described [14] (link). A goat anti-mouse (GAM) IgG was used as an isotype-matched control in all the experiments. For immunofluorescence experiments, an Alexa Fluor 546-conjugated donkey anti-mouse IgG (h4l) was used (Invitrogen Molecular Probes, Carlsbad, CA, USA). Nitric oxide levels were measured using a commercial Griess reagent kit (Promega, Madison, WI, USA).