Fungal Immune Response Protocol

Luminol, Lucigenin, Isoluminol, HRP Type VI, PMA, SOD from bovine erythrocytes and zymosan were obtained from Sigma (St Louis, MO). FCS, HBSS, H₂DCF-DA were from Invitrogen Molecular Probes (Oregon). DMEM was purchased from PAA (Vienna, Austria), anti-mouse antibodies CD16/CD32, CD11b-FITC, CD11c-APC, F4/80-PE-Cy5 were obtained from BD Bioscience (Mountain View, CA). Rich medium (YPD) and synthetic complete were prepared essentially as described (Kaiser et al., 1994 ). BMDM media are composed of DMEM, 10% heat-inactivated FCS, 20% l-conditioned medium. mDC media are composed of DMEM, 10% heat-inactivated FCS, 10% X-conditioned medium. C. albicans strains were grown at 30°C in YPD medium overnight, diluted to an OD₆₀₀ = 0.2 the next morning, grown to the logarithmic growth phase and used for the experiment unless indicated otherwise. For the preparation of mature filaments, an overnight culture of C. albicans was diluted 1:10 in YPD + 10% FCS and grown at 37°C for 3–4 h. For experiments requiring stimulation of macrophages with filaments, an aliquot of each culture was pelleted and the dry weight was determined by routine procedures. Aliquots of cultures equalling the indicated dry weights of yeast or filaments were used for experiments. Typically, 4 × 10⁴ yeast cells correspond to 1 μg dry weight.