Experiments and precultures were performed in sterile RPMI 1640 medium without l- glutamine (Quality Biological, Gaithersburg, MD) containing 165 mM MOPS (morpholinepropanesulfonic acid [Sigma-Aldrich]) at pH 7.0. Fresh cultures from frozen stocks were shaken overnight in RPMI at 37°C and 5% CO2, rinsed, enumerated, and diluted in RPMI-MOPS. Ferritin from equine spleen (Sigma-Aldrich), human apo-transferrin (Sigma-Aldrich), and/or iron (EMD Millipore), and C. neoformans or C. gattii (final concentration of 80,000 per ml) in RPMI-MOPS with 1 μM iron were arrayed in a 2-ml-deep-well plate in a final volume of 0.4 ml/well. The culture plate was sealed with a gas-permeable membrane (USA Scientific, FL) and shaken at 37°C and 5% CO2 for 24 h. Fungal growth and inhibition were determined by dilution plating for CFU. Data were normalized to a percentage of the control by the following formula: 100 × (experimental no. of CFU)/(control no. of CFU). The control conditions used to normalize each experiment are indicated in the legend to Fig. 8 .
Gas permeable membrane
Manufactured by USA Scientific
A gas-permeable membrane is a thin, semi-permeable barrier that allows the selective passage of gases while restricting the flow of other substances. This membrane is designed to control the exchange of gases, such as oxygen, carbon dioxide, and other specific gases, between two environments.
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2 protocols using gas permeable membrane
1
Fungal Growth Inhibition Assay
2
Quantifying Bacterial Biofilm Formation
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The mass of biofilms was assessed using Crystal Violet (CV) staining (0.1%) [41 (link)] and chemiluminescence intensity measurements as an additional determinant of biofilm viability [42 ]. In each experiment an overnight culture of P. aeruginosa Xen 5, P. aeruginosa PAO1, P. aeruginosa P14 in TSB or S. aureus Xen 29, B. subtilis ATCC6051, E. coli MG1655, E. coli RS218 and C. albicans 1409 in LB was diluted to ~ 105 CFU/ml. When required, bacterial suspensions were placed on glass slides coated with poly-Lysine/F-actin or in flat bottom PVC microplates (MP Biomedicals; Solon, OH) sealed with a gas permeable membrane (USA Scientific; Ocala, FL). Bacteria or C. albicans were grown in 50% LB broth with or without F-actin, DNA, NFs, vimentin or Pf1 bacteriophage. A stationary biofilm was allowed to form for 24 or 48 h. PA Xen5 and S. aureus Xen29 chemiluminescence signals were evaluated using a Fuji Film LAS-300 system. Densitometry analysis was performed using Image Gauge (version 4.22) software.
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