C. elegans Hypoxia Survival Assay

C. elegans Bristol-N2 was maintained at 20°C until adult stage on nematode growth medium (NGM) plates. To collect eggs, fertilized C. elegans adults were incubated with alkaline lysis buffer while being shaken for 20 min at room temperature and subsequently washed twice with M9 buffer. To arrest the larvae to L1 stage, the C. elegans eggs were transferred for hatching on new NGM plates and incubated at 20°C for 20 h without food. L1 synchronized worms were transferred to fresh NGM with nonpathogenic E. coli OP50 for 48 h to mature up to the L4 stage. Five worms per well were then distributed on 24-well plates with NGM agar with 40 μM 5-fluoro-2′-deoxyuridine (FUDR) (Sigma) to prevent progeny production (55 (link)). Additionally, 500 μl HBSS medium (Lonza) was added to each well. Sets of five wells either contained 2 × 10⁸E. coli OP50 cells/well or 3 × 10⁵C. albicans SC5314 cells/well. Plates were incubated at 25°C in normoxic and hypoxic (1% O₂) cell incubators with 5% CO₂ for 48 h, and C. elegans survival was subsequently recorded with a Nikon SMZ1500 stereomicroscope.