Scheme of the established solid medium based dietary restriction method to apply dietary restriction during development (dDR) in <i>C. elegans</i>.

<p><i>Escherichia coli</i> (<i>E.</i> coli) OP50, serving as food source for <i>C. elegans</i>, was cultivated at 37°C until an optical density (OD₆₀₀) = 1.5 was reached (OD₆₀₀ = 1.0≈1.0×10⁹ cfu/ml). Different ODs (6.0, 3.0, 1.5, 1.0, 0.7, 0.3) were adjusted in M9 buffer. Standard Nematode Growth Medium (NGM) was used for <i>ad libitum</i> (AL) condition. For dietary restriction (dDR), bactopeptone was omitted from NGM agar plates to prevent bacterial growth. A defined amount (250 µl) of different OP50 suspensions was seeded on dDR plates, resulting in a gradient of colony forming units (cfu) per plate. For AL plates, an OP50 suspension of OD1.5 was used. Nematodes were synchronized by hypochlorite treatment, and 500 eggs per agar plate were sorted via cytometry-based COPAS Biosort system. Animals were cultivated at 20°C and harvested at second larval stage (L2, 24 h after hatching), fourth larval stage (L4, 48 h) or after reaching adulthood (adult, 72–78 h). At L4 stage, nematodes were transferred once to fresh agar plates.