Procartaplex panels

A. fumigatus strains AF10 (clinical isolate 90240; ATCC) or AF293 (MYA-4609, ATCC; obtained from Nancy Keller, University of Wisconsin) were grown on minimal glucose media (MGM) plates using an overlay method for 3 days at 37°C (22 (link)) and conidia were harvested in PBS by gentle scraping with a cell spatula, passed through two layers of sterile Miracloth and a 40μM strainer (39 (link)). Conidia were enumerated using a hemocytometer and numbers subsequently verified by plate culture for CFU. Mice were anesthetized by IP ketamine and challenged IN with indicated doses of conidia in 25μl of sterile PBS. In some experiments, mice received 200mg/kg freshly prepared cortisone acetate IP on day −3, 0 (day of challenge) and day 4. Mice were euthanized at the indicated times and 1mL BAL collected for cell counts, cytospins for leukocyte differential and colony forming unit (CFU). The left lung was processed for histology and staining with H&E and with Gomori methamine silver (GMS) (39 (link)). Images were captured with a NanoZoomer digital slice scanner and NDP.view2 software (Hamamatsu Photonics, Japan). The right lung was homogenized in 500 μl PBS. For fungal burden, aliquots of BAL and lung homogenates were each plated on MGM for CFU analysis. ELISAs on BAL samples were performed using ProcartaPlex panels from Thermo Fisher Scientific.