TITLE: Fluorescent caspase-3 staining to assess induction of apoptosis in A6 cells (Plos One)
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.
?. Seeding A6 cells:- Detach A6 cells from a cell culture flask using trypsin- Centrifuge for 5 min at 1500 rpm- Wash the cells with 70% HBSS- and in meantime count the cells- Centrifuge for 5 min at 1500 rpm - Resuspende 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 A6 cells attach for 2 hours at 26°C and 5% CO2- Afther 2 hours, 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.
?. Fixation and staining of apoptotic cells: (Work in the dark)  - Gently wash the infected cells 2 times with 70% HBSS+- Fix the infected cells with 0.5 mL of 3% paraformaldehyde for 10 min - Wash 2 times with 70% HBSS+- Permeabilise with 200 µL of 0.1% trition in 70% HBSS+ on room temperature during 2 min.- Wash 2 times with 70% HBSS+- Incubate 1 hour with a primary antibody (1/1000):            * Anti-caspase-3 antibody produced in rabbit -> Sigma-Aldrich- Wash 3 times with 70% HBSS+- Incubate for 1 hour with a secondary antibody (1/500):        * Alexa Fluor 568 goat anti-rabbit IgG- Wash 3 times with 70% HBSS+- Incubate with Hoechst for 15 min- Wash 3 times with 70% HBSS+- Mount the coverslips using ProLongGold antifade mountant- Use fluorescence microscopy to analyse apoptosis
?. Bd infection of A6 cells:- Isolate Bd zoospores and spin them down for 5 min at 3000 rpm (20°C)- Remove the supernatant, resuspend them in cell medium B and count the spores- Dilute the spores to a concentration of 10e6 spores/mL in cell medium B- Add 1 mL of the spores suspension (= 10e6 spores) to the wells containing A6 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 the cell medium B with cell medium A for 4 to 6 days (depending on the experimental setup) at 20°C, 5% CO2IMPORTANT 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.