Killing Assays

Killing assays were performed in 96‐well clear‐bottom plates. A total of 5 × 10⁴ human neutrophils were plated with C. albicans at a multiplicity of infection (MOI) of five in 100 μL of complete RPMI. The plates were incubated at 37°C and 5% CO₂ for 2 hours. Following the initial incubation, mammalian cells were lysed with 4‐times NP40, and each well received an addition of optimized yeast growth media (MOPS‐RPMI) to supplement Candida growth. Finally, 10% PrestoBlue Cell Viability Reagent (Thermo Fisher Scientific) was added to each well. The plates were then incubated at 35°C and 5% CO₂ until percent remaining live pathogen was measured at a predetermined endpoint in a plate reader (SpectraMax i3x; Molecular Devices, Sunnyvale, CA). Results were reported as the percent of pathogens killed by the neutrophils (“percent killing”).

To determine the effects of patient serum on neutrophil function, plasma samples were thawed and chelated with CaCl₂ (20% m/v) at a 1:100 ratio to remove EDTA (as EDTA was observed to kill C. albicans independently) and convert plasma to serum. Healthy control neutrophils were incubated at 37°C and 5% CO₂ with Candida at a MOI of 10 for 2 hours in RPMI containing 20% serum from healthy control and patients with cirrhosis. Mammalian cell lysis and percent remaining live pathogen was measured as described previously.

To determine the medication effects on neutrophil function, 5 × 10⁴ human neutrophils were plated in 100 μL of complete RPMI media containing varying concentrations of drug and incubated at room temperature for 5 minutes. C. albicans was then added to wells at a MOI of 10. The plates were incubated at 37°C and 5% CO₂. Following the initial incubation, cell viability was confirmed with AO/PI to ensure the drugs were not toxic to the neutrophils. Then, mammalian cells were lysed, and the percent remaining live pathogen was measured as described previously.