Dichlorofluorescin diacetate (DCFDA) is a popular fluorescence-based probe for reactive oxygen species (ROS) detection in vitro and in vivo, and has been used for this purpose in C. elegans. DCFDA is first deacetylated by endogenous esterases to dichlorofluorescein (DCFH), which can react with several ROS to form the fluorophore DCF (reviewed by Halliwell and Gutteridge, 2007). The DCFDA assay in C. elegans is sometimes performed using lysed worms, following high intensity sonication. This process disrupts the outer cuticle and internal membranes, causing intracellular, as well as intraorganelle, contents to be released. Worm lysis will cause the release of transition metal ions such as iron. Free iron may participate in redox cycling to generate ROS, for example, by Fenton chemistry (Halliwell and Gutteridge, 2007). Therefore, apparent ROS levels detected in lysed worms may be un-physiological. However, DCFDA readily diffuses into cells where it undergoes deacetylation, and this allows ROS measurement in whole worms. If whole animals are utilized, cells are not disrupted and iron remains sequestered.

Using DCFDA and a synchronized worm cohort, we measured the amount of ROS in equivalent numbers of lysed and whole worms. The gradient of the linear regression curve (expressed as ΔRFU/Δmin) (Figure 1) is the rate of fluorescent DCF production. This gradient can be, very approximately, converted to the rate of H2O2-equivalent ROS production (nmol/min) by spiking known amounts of H₂O₂. It should, however, be noted that H₂O₂ itself does not oxidize DCFH, and hence, the free radical reactions resulting in increased DCF signal following H₂O₂ spiking are likely complex. Therefore, the calibration ratio can only be considered to provide an approximate estimation of ROS produced. Nevertheless, we found that the rate of H₂O₂-equivalent production was more than an order of magnitude faster in lysed (4.1×10-3nmol/min/worm) compared to whole animals (3.6×10-4 nmol/min/worm). Using the O₂ consumption rate measured in the same worm cohort (0.018 nmol/min/worm), the % of O₂ consumed leading to ROS generation can be estimated by dividing the rate of H₂O₂-equivalent production by the rate of O₂ consumption. Using this approach, we found apparent ROS production to be around 23% for lysed worms, but only 2% for whole worms. Current estimates of in vivo mitochondrial ROS production range from 0.2 to 2% (Balaban et al., 2005). Assuming all ROS measured using the DCFDA assay to be of mitochondrial origin, ROS production is clearly overestimated by a factor of at least 10-fold, and may be as much as 100-fold, in lysed worms compared to whole worms. Therefore, when using the DCFDA assay, whole worms should be utilized instead.

C. elegans is greatly suitable in suppressor screens and has strong potential to screen for the effects of modifiers at the subcellular level as it is transparent at all stages of lifespan. Here, we describe a 96-well plate assay that illustrates how high resolution imaging may be used in day-to-day research as well as RNAi and drug screens. We used the Plate Runner HD® (Trophos, France), a 96/384-well plate motorized device able to collect fluorescence at resolutions ranging from 1024×1024 px (1 px is 7.4 µm) to 8192×8192 px (1 px is 1 µm). This device has high depth of field (40 µm at resolution of 7.4 µm; 8 µm at resolution of 1 µm), thus allowing fluorescent signals to be quantified from whole animals. This device also has a wide-field objective that allows single images of the whole well to be acquired at once (Fig. 1A). To develop drug screening for neuromuscular disease, we used C. eleganstransgenics that co-express GFP and the oculopharyngeal muscular dystrophy (OPMD) protein PABPN1 in body wall muscle cells. Mutant PABPN1 animals show defective motility accompanied by loss of GFP nuclei (20 signals lost in 3-day adults) and muscle cell degeneration (Catoire et al., 2008). These phenotypes are aggravated by sirtuin (sir-2.1/SIRT1) activation and ameliorated by sirtuin inhibition (Catoire et al., 2008; Pasco et al., 2010). Resveratrol, an indirect SIRT1 activator, enhances mutant PABPN1 toxicity whereas EX-527, a selective SIRT1 inhibitor, has the opposite effect (Catoire et al., 2008; Pasco et al., 2010). We used these chemicals to normalize a drug screen assay (Fig. 1B). Synchronised L1 larvae are incubated at 20°C with bacteria and drugs (40 L1 in 50µl/well; 5 wells/point) until they reach adulthood. At 46 hours, FUDR (0.1 mg/ml) is added to prevent egg-laying and hatching. Day-3 adults are immobilized using 2,3-butanedione monoxime (0.1 M, 300 µl/well). Morphometric analysis of images (here 2048×2048) is performed using Image J and a script developed in the laboratory. Average numbers of GFP signals/worm are integrated into a database and subjected to statistical analysis. Drug screening is now ongoing using this assay. Perfect immobilization of animals is required for imaging at 4096×4096 and up. Similar strategies may be used in the development of a variety of suppressor screens.

In the past decade C. elegans has become a popular model in investigating molecular effects of phytochemicals. Among these, flavonoids represent a major group of plant secondary metabolites shown to alter lifespan and stress resistance in C. elegans ( Kampkoetter et al., 2008). However, bioavailability of these compounds is rather low. Detection in worms usually requires analytical techniques such as HPLC and rather large amounts of worm material but gives no information on where in worms the compound accumulates (Kampkoetter et al., 2008). Here we report a method to detect and semi-quantify flavonoids in C. elegans by analysis of the fluorescence after derivatisation in vivo. For this purpose, we used 2-aminoethyl diphenyl borate (Naturstoff reagent A (NSRA)), which is employed in thin layer chromatography, plant histology and cell culture to enhance auto-fluorescence of plant polyphenols (Neu, 1956; Ernst et al., 2010). NSRA forms a chelate with the polyphenolic compounds, leading to a shift of the spectral band to a longer wavelength (bathochromic shift) and to an intensified signal.

We exposed wild type N2 L4-larvae on NGM plates to the flavonoids myricetin, quercitin or kaempferol in increasing concentrations (0, 10, 50 or 100 µM) for 48 hours. Thereafter, the worms were incubated in M9 buffer containing 10% heat-killed OP50 and 0.2% NSRA for 2 hours. Subsequently, fluorescence was monitored using a confocal laser scanning microscope with excitation at 488 nm and emission at 590-620 nm (myricetin and quercitin) and at 540-560 nm (kaempferol).

NSRA selectively increased the fluorescence of the flavonoids, making them detectable in C. elegans. Highest fluorescence was always obtained in gut epithelial cells (A) as the sites of flavonoid uptake and intensity reflected the flavonoid concentrations in a dose-dependent manner (B). This shows that NSRA is an easy-to-use and cheap tool to visualize and semi-quantify flavonoids in vivo in C. elegans.