Steady glow

The ability of NPD6433 to rescue human cell growth in co-culture with a GFP-expressing strain of C. albicans SN95 alone and in combination with fluconazole was assessed as previously described.^{79 (link)} Luciferized 293T cells were seeded at 1 × 10⁵ cells/mL X 20 μL in DMEM medium containing 10% FBS and incubated overnight at 37°C in 5% CO₂ in a 384-well plate. The following day, 20 μL of DMEM medium alone or inoculated with 5 × 10³ cells/mL exponential phase C. albicans cells was added to the appropriate wells. A Tecan D300e compound dispenser was used to add DMSO-based NPD6433 or fluconazole to each well at the indicated final concentrations. Co-cultures were incubated for 24 h at 37°C in 5% CO₂. Fungal growth was measured by reading the GFP fluorescence signal (480 nm excitation and 540 nm emission). Mammalian cell growth was measured by adding 5 μL Steady-glow (Promega) to each well, incubating for 15 min, and reading luminescence on a Tecan Infinite 200 Pro. For each species, results were normalized to the growth of that species in monoculture in the same plate under the same conditions. All experiments were performed in technical quadruplicate and biological duplicate and results plotted in GraphPad Prism.