Flat bottomed microtiter plate

Spores (OD600 =  ~ 1, in MIlliQ water) were heated for 5 h at 40, 50, 60, 65, 70, 75 or 80 °C, followed by cooling in a water–ice bath (≥15 min). Spores (OD₆₀₀ =  ~ 1, 150 µl/well) were added to a 96-well flat-bottomed microtiter plate (black wall, Greiner Bio-One), and the optical density and fluorescence intensity were measured by a BioTek plate reader. Data were collected from at least two independent tests, each of them had three biological repeats.

BAL and PL suspensions were centrifuged for 10 min at 410 × g. The cells were resuspended in 1.0 mL of RPMI-1640 (Nutricell, Campinas, Brazil) supplemented with 10% heat-inactivated fetal calf serum (Nutricell), penicillin (100 UI mL^-1), streptomycin (100 mg mL^-1; Sigma-Aldrich, St. Louis, MO, USA), and amphotericin B (0.25 μg mL^-1; (Sigma-Aldrich). The cell concentration was adjusted to 1.0 × 10⁵ mononuclear phagocytes mL^-1, as judged by the uptake of 0.02% neutral red (Sigma-Aldrich) and confirmed by the expression of F4/80 by fluorescence-activated cell sorting. Cells were plated in 96-well flat-bottomed microtiter plates (Greiner BioOne, Frickenhausen, Germany) and incubated for 2 h at 37°C in a 5% CO₂ atmosphere in a humidified chamber to allow cells to adhere and spread. Non-adherent cells were removed by washing the wells three times with RPMI. The remaining adherent cells, which comprised > 95% mononuclear phagocytes as assessed by morphological examination, were used for the experiments. The adherent cells were cultured at 37°C and 5% CO₂ in supplemented RPMI-1640 with or without heat-killed spores of R. oryzae (R. oryzae-Ag) at a spore:cell ratio of 1:1. As an internal control for macrophage activity, the cells were cultured with 10 μg ml-1 lipopolysaccharide (Sigma-Aldrich). After 24 h, cell-free supernatants were harvested and stored at -80°C for cytokine analysis.