For example, the relative positions of spores and macrophages allow quantifying the amount of spores that buy SB203580 adhere to macrophages and/or aggregate to each other forming clusters of spores. The method is suitable to elicit the comparison of the 28 human pathogenic species of the
zygomycetes.[18] Two strains of L. corymbifera JMRC:FSU:9682 (virulent) and JMRC:FSU:10164 (attenuated) were cultivated on KK1 medium (10 g l−1 glucose, 3 g l−1 KH2PO4, 1.25 g l−1 K2HPO4, 0.5 g l−1 MgSO4 × 7H2O, 2 g l−1 yeast extract, 1 g l−1 KNO3, 0.5 g l−1 KCl, 4.4 g l−1 NaCl) at 30 °C for 5–7 days. Sporangiospores were freshly harvested from the sporangia grown on the KK1 agar plates and collected in sterile phosphate buffered saline (PBS). For 10× PBS (stock solution) we dissolved 80 g NaCl, 2 g KCl, 26.8 g Na2HPO4 × 7H2O and 2.4 g KH2PO4 in 800 ml LDE225 clinical trial H2O, adjusted
to pH 7.4 with HCl and supplemented to the volume to 1 litre with distilled water to gain a 10× concentrated stock solution. The 10× PBS was diluted 1 in 10 for the final working solution concentration. Spores were labelled with 0.1 mg ml−1 FITC (Fluorescein isothiocyanate; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in 0.1 mol l−1 Na2CO3 at 37 °C for 45–60 min. Those spores were washed three times with PBS and finally resuspended in RPMI-1640 (BE12-167F; Lonza, Verviers, Belgium) fluid cell culture medium.[19] Concentration of spores was determined using a Thoma chamber: area 4 × 10−2 mm, depth 0.1 mm; volume = 4 × 10−6 ml. Spore suspensions subjected to the phagocytosis assays immediately are referred to as resting spores. Swollen Bay 11-7085 spores were obtained after incubation of spore suspensions for 3 h and under constant gentle shaking at 37 °C. Opsonised spores are resting spores that were resuspended in serum from clotted human male whole blood (Sigma) instead
of RPMI-1640 and incubated for 30 min under constant gentle shaking at 37 °C to recruit the opsonin molecules to the spore surface, which are enhancing phagocytosis by marking the fungal spores for attraction, ingestion and destruction by phagocytes as part of the innate immune response (opsonisation). Murine alveolar MH-S macrophages (ATCC: CRL-2019) were cultivated in RPMI-1640 supplemented with 10% heat inactivated foetal bovine serum (ATCC-30-2020), 1% ultraglutamine 1 (17-605E/U1; Lonza) and 550 μl l−1 (50 mg ml−1) gentamicin sulphate (17-518Z; Lonza) at 37 °C in 5% CO2. For phagocytosis assays, macrophages were seeded on glass cover-slips in 24 well plates (NUNC, 142475) at a density of 2 × 105 cells per well to adhere overnight. Macrophages were labelled with Vybrant DiD (Life Technology GmbH, Darmstadt, Germany) prior to the experiment.
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