Modified Dietary Restriction Assay for E. coli

The msDR method was modified from previously described [49] (link). Overnight culture of E. coli OP50 grown at 37°C was centrifuged at 3,000 rpm for 30 minutes to collect bacteria cells. The bacterial pellet was washed with the S buffer, and the bacterial concentration was adjusted to 1.0×10¹² cfu/ml. Serial dilutions were performed to achieve bacterial concentrations of 1.0×10¹¹, 1.0×10¹⁰, 1.0×10⁹, and 1.0×10⁸ cfu/ml. Diluted bacterial cultures were spotted onto DR agar plates, which were modified from the standard nematode growth media (NGM) plates by excluding peptone and increasing agar from 1.7% to 2.0%. Carbenicillin (50 µg/ml) was added to the agar plates to further prevent bacteria growth. Synchronized L4 larvae growing under standard lab conditions (NGM plates with OP50 food, 20°C) were transferred to fresh NGM plates with OP50 food and 5 µg/ml of FUdR, and were incubated at 25°C overnight. Day 1 adult animals were then transferred to DR agar plates seeded with OP50 at different concentrations.
In the first week of lifespan experiments and heat stress assays, 5-fluorodeoxyuridine (FUdR) at 50 µg/ml was also added into the agar plates to prevent progeny from hatching.