Methods Fungal Strains and culture conditions Candida parapsilosi

Methods GDC-0994 chemical structure Fungal Strains and culture conditions Candida parapsilosis GA1 and lipase deficient (ΔCplip1-ΔCplip2/ΔCplip1-ΔCplip2::FRT) strains [13] were

maintained at -80°C in 35% glycerol. If not mentioned otherwise, the cells were grown in YPD (1% yeast extract, 2% bactopeptone, 2% glucose). Monocyte isolation and dendritic cell differentiation Human Selleckchem Adriamycin peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat blood samples from healthy donors by Ficoll Paque Plus (GE Healthcare) density gradient centrifugation. Monocytes were isolated by adherence on tissue culture plastic plates. Immature dendritic cells were prepared by culturing monocytes for five days with 1000 U/ml human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF; Sigma) selleck chemicals llc and 1000 U/ml human recombinant interferon-α (IFN-α; Sigma) in RPMI-1640 medium (Gibco) complemented with 10% heat-inactivated FBS (Gibco) and 1% penicillin/streptomycin solution (Gibco) in 6-well tissue culture plate (Sarstedt). Mature dendritic cells were obtained from immature dendritic cells by stimulation with 10 ng/ml recombinant TNFα (R&D Systems) for 24 hours. In vitro infection For infections, iDC and mDC cells were co-incubated with C. parapsilosis cells at effector-to-target ratios of 1:5 in six-well plates. Samples were incubated for various time at 37°C and 5% (v/v) CO2. For gene expression studies DCs were harvested after 1 h and 24 h co-incubations,

for cytokine measurement supernatants were collected after 24 h and 48 h. Killing assays Co-cultures of the DCs and C. parapsilosis were performed according to our described protocol [13] with some acetylcholine modifications. Briefly, C. parapsilosis cells were grown overnight, washed three times in PBS, counted using a hematocytometer, and suspended in RPMI-1640 medium (Gibco). The cells were then co-incubated with DCs as described above. As a control, the same number of C. parapsilosis cells were inoculated in the RPMI-1640 medium (Gibco) complemented with 10% heat-inactivated FBS (Gibco) and 1% penicillin/streptomycin solution (Gibco)

with no effector cells. The wells were then incubated at 37°C for 3 h, and washed three times with PBS to remove nonadherent Candida cells. Yeast cells were liberated from DCs by forcibly disrupting the DCs through pipetting them in distilled water for 2 min. The yeast cells were collected, counted, and serially diluted prior to being plated. Cells were plated in YPD agar and incubated for 3 days at 30°C. The killing efficiency was calculated by normalizing the number of CFU (colony forming unit) counted from the DC infected wells to the total number of CFU of C. parapsilosis detected from the control wells, and multiplied by 100 for percentage. Phagocytosis assays Infections were performed as described above and the phagocytosis was monitored by fluorescent microscope after 1 h of co-incubation. Briefly, DCs were treated with FITC-labeled C.

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