Computational protocol: Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue

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Protocol publication

[…] Human lung A549 epithelial cells (ATCC CCL-185) were grown using DMEM supplemented with 10% fetal bovine serum (FBS), antibiotics (100 U ml−1 penicillin and 100 mg ml−1 streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5% CO2. A549 cells were regularly passaged in T-75 cell culture flasks. A. fumigatus was grown on standard Vogel's agar at 37 °C for 5 days. Conidia were collected using 0.05% Tween 80, re-suspended in 20% Vogel's liquid medium and incubated for 12 h at 25 °C. For co-cultures, human lung epithelial cells were plated on glass chamber slides Lab-Tek II (Nunc) 2 days before imaging and incubated for 16 h with A. fumigatus conidia reaching 75–90% confluence on the day of the experiment. For imaging experiments, cells were incubated for 15 min at 37 °C with compounds 5–8 (5 μM for compounds 5–7 and 2 μM for compound 8) and imaged without washing in phenol red-free DMEM under a Zeiss LSM 510 META fluorescence confocal microscope equipped with a live cell imaging stage. Fluorescence and bright-field images were acquired using × 40 or × 63 oil objectives. Fluorescent probes were excited with 488 nm (compounds 5–8) or 543 nm (Syto82) lasers. Confocal microscopy images were analysed and processed with ImageJ. Quantitative analysis of mean fluorescence intensities in competition experiments was performed with Imaris by calculating the mean intensity of each hyphae as independent regions of interests. For competition assays, all images were acquired and analysed using exactly the same conditions. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Aspergillus fumigatus, Homo sapiens, Fungi
Diseases Mycoses
Chemicals Clomiphene