D300e compound dispenser

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.

Human cell line 293T (female, HEK 293T Cat# CRL-3216) was obtained from the American Type Culture Collection (ATCC). Culture medium consisted of high glucose Dulbecco’s-Modified Eagle Medium (DMEM, Invitrogen Cat#11965092) containing 2 mM glutamine and 10% heat-inactivated fetal bovine serum (FBS, Sigma). Cultures were grown at 37°C under 5% CO₂. Low passage stocks were banked in liquid nitrogen and experiments performed within 12 serial passages from thawing. Stocks were confirmed negative for mycoplasma contamination by PCR-based testing.
For cytotoxicity assays, cells were seeded at 2,000 cells/well in DMEM medium containing 10% FBS and incubated overnight at 37°C in 5% CO₂. A Tecan D300e compound dispenser was used to add DMSO-based compounds to each well at the indicated final concentrations. Cells were incubated for a further 48 hours at 37°C in 5% CO₂. Human cell metabolic activity was measured by adding a 1:20 dilution of Alamar blue, incubating under the same conditions for 3 additional hours, and reading fluorescence on a Tecan Infinite 200 Pro (Ex 560/ Em 590 nm). All experiments were performed in technical triplicate and biological duplicate.