Measuring Worm Autofluorescence as Aging Marker

We recently developed a method to measure autofluorescence as an aging marker for individual worms (28 (link)). In brief, 6-day-old worms fed OP50 or FC from 0 days were washed with M9 buffer and placed into 1.0 μL of M9 buffer on a 384-well black plate (Stem, Tokyo, Japan) covered with Saran Wrap (Asahi Kasei, Tokyo, Japan). The blank (M9 buffer only) data were checked three times for each well, considering the fluctuation across wells. Minimal detection limits and quantifiable limits were determined on the basis of blank data on each day as μ (mean of the blank) + 3.29σ (standard deviation) and μ + √2 × 10σ, respectively. The autofluorescence in the worm body was captured using a multimode grating microplate reader model SH-9000Lab (excitation, 340 nm; emission, 430 nm; Corona Electric, Ibaraki, Japan). After measurement, each worm was individually maintained on a 4-cm-diameter plate covered with OP50 or FC (2 mg/10 μL M9 buffer) at 25°C. The data on worms that died in 2 days were excluded because the autofluorescence could be due to death and not from AGEs (62 (link)). The assay was performed with more than 20 worms. Three biological replicates were analyzed for this study.