TITLE: Fluorescent in vitro model to assess adhesion and invasion of Bd in PAK 
AUTHORS: Elin Verbrugghe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The largest current disease-induced loss of vertebrate biodiversity is due to chytridiomycosis and despite the increasing understanding of the pathogenesis, knowledge unravelling the early host-pathogen interactions remains limited. </span><span style = "font-style:italic;">Batrachochytrium dendrobatidis </span><span style = "font-style:italic;font-style:italic;">(</span><span style = "font-style:italic;">Bd</span><span>)zoospores attach to and invade the amphibian epidermis, with subsequent invasive growth in the host skin. Availability of an</span><span style = "font-style:italic;"> in vitro</span><span> assay would facilitate in depth study of this interaction while reducing the number of experimental animals needed. We describe a fluorescent cell-based </span><span style = "font-style:italic;">in vitro</span><span> infection model that reproduces host-</span><span style = "font-style:italic;">Bd</span><span> interactions. Using primary keratinocytes from </span><span style = "font-style:italic;">Litoria caerulea</span><span> and the epithelial cell line A6 from </span><span style = "font-style:italic;">Xenopus laevis</span><span>, we reproduced different stages of host cell infection and intracellular growth of </span><span style = "font-style:italic;">Bd</span><span>, resulting in host cell death, a key event in chytridiomycosis. The presented </span><span style = "font-style:italic;">in vitro</span><span> models may facilitate future mechanistic studies of host susceptibility and pathogen virulence. </span></div></div>

[STEPS]
?. Prepare Cell Medium A: L15 medium: 70%Distilled water: 20%Fetal bovine serum 10%
?. Prepare Cell Medium B: L15 medium: 40%Distilled water: 55%Fetal bovine serum: 5%
?. Coat coverslips with Rat tail collagen: Add glass coverslips in a 24-well tissue culture plate. Coat the glass coverslips at 37°C for 2 hours. Therefore, carefully aspirate the Collagen Coating Solution and add 200 µl per well (so per coverslip). After 2 hours, rinse the coated surface twice with HBSS-. Coated tissue culture ware may be used immediately or air-dried and stored at 4 oC for up to one week.
?. Staining of Primary Keratinocytes (PAK): (Work in the dark)- Add 1 ml of 3 µM Celltracker green CMFDA in cell medium A to the PAK - Resuspend carefully- Incubate for 30 min at 20°C 5% CO2- Centrifuge for 5 min at 1500 rpm and 20°C- Resuspend the pellet in 70% HBSS- and in meantime count the cells- Centrifuge for 5 min at 1500 rpm - Resuspend the cells in cell medium A to reach a concentration of 10e5 cells per mL- Seed the cells at a concentration of 10e5 cells per well (so add 1 mL) which contains a collagen-coated glass coverslip- Let the PAK attach for 1 hour at 20°C and 5% CO2- Afther 1 hour, wash the cells with 70% HBSS+- The cells are now ready to be exposed to Bd sporesIMORTANT NOTE: Include a control well that can be used to check the cells throughout the entire protocol via light microscopy.
?. Staining of Bd zoospores: (Work in the dark)  - Isolate Bd zoospores and spin them down for 5 min at 3000 rpm (20°C)- Remove the supernatant and resuspend them in 3 µM celltracker red CMPTX solution for 45 min at 20°C. Important here is that the 3 µM celltracker solution should be diluted in CELL MEDIUM B in order to ensure the motility of the zoospores. - After 45 min, spin the spores down for 3 min at 3000 rpm- Resuspend them in cell medium B and count the spores- Dilute the spores to a concentration of 10e6 spores/ml in cell medium BIMPORTANT NOTE: Bd zoospores lose their motility when exposed to cell medium A. IMORTANT NOTE: Include a control well that can be used to check the motility of the spores throughout the entire protocol via light microscopy.
?. Bd infection of PAK cells: (Work in the dark)- Add 1 ml of the spore suspension (= 10e6 spores) to the wells containing celltracker-labelled PAK cells (on a coverslip). As such the spores are seeded at a MOI of 10:1. - Incubate for 2 hours at 20°C 5%CO2.- After 2 hours gently wash the infected cells three times with 70% HBSS+ to remove non-adherent spores- Replace cell medium B with cell medium A for another 2 hours to assess ADHESION or 22 hours to assess invasionIMPORTANT: Use the control well to check the adhesion/invasion of the spores to/in the cells via light microscopy.
?. Visualisation of early Bd-PAK interactions: (Work in the dark)ADHESION: 4 hours post infection (2 hours with cell medium B and 2 hours with cell medium A)INVASION: 24 hours post infection (2 hours with cell medium B and 22 hours with cell medium A)- Wash the infected cells 3 times with 70% HBSS+- Fix the infected cells with 0.5 mL of 3% paraformaldehyde for 10 min - Stain the nulear content with Hoechst- Wash 2 times with 70% HBSS+- Mount the coverslips using ProLong Gold antifade mountant- Use fluorescence microscopy and confocal laser scanning microscopy to analyse Bd-PAK interactions