C elegans experiments

2.2.1 Strains growth and maintenance

The C. elegans strains N2 (wild isolate), and daf-16(mu86) (10980 bp deletion), hif-1(ia4) (1231 bp deletion), trpa-1(ok999) (1334 bp deletion), egl-9(sa307) (243 bp deletion) and egl-

9(n586) (base substitution) were acquired from the Caenorhabditis Genetics Center (CGC),

University of Minnesota. All strains were cultured on solid nematode growth media (NGM) agar (Table 2.1).

Table 2.1: Recipe for making NGM agar.

Reagents For 100ml of dH2O Agar 1.7g NaCl 0.3g Peptone 0.25g Autoclave 1M CaCl2 100l 1M MgSO4 100l 1M KPO4 2.5ml Cholesterol (5mg/ml in Ethanol) 100l Nystatin (10mg/ml in DMSO) (N6261) 100l

FuDR (150mM) 33l (lifespan assay plates only)

10ml of NGM agar was poured into each 55mm plate and allowed to solidify and dry at room temperature overnight. Once dry, NGM plates were seeded with 200l of Escherichia Coli (E.

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cultures were seeded from a single colony grown in LB broth overnight at 37°C in a shaking incubator. C. elegans strains were maintained in an incubator (RLSD01502; LabCold) at 20°C unless stated otherwise. Strains were viewed using a stereomicroscope (Motic SMZ-171T) and were handled using a flame sterilised platinum worm pick (#59-AWP; WormStuff, Genesee Scientific).

2.2.2 C. elegans lifespan assays

NGM plates used for lifespan assays were additionally supplemented with 50M (33l of 150mM in 100ml of NGM agar) of 5-Fluoro-2’-deoxyuridine (FuDR; F10705-1.0; Melford Laboratories) to prevent growth of unwanted offspring. Lifespan assays were conducted on a population of synchronised worms. Adult worms (containing eggs) were placed into a drop (~10l) of alkaline hypochlorite solution (250l of dH2O, 230l of sodium hypochlorite and 30l

of 10M NaOH) on the NGM plate. The solution killed the worms, causing eggs to be released. Hatching occurs simultaneously resulting in a synchronised population. Offspring were grown and at least 50 L4 stage worms were transferred onto new NGM plates containing FuDR for lifespan assays. Worms were transferred onto new plates weekly but more frequently if the seeded OP50 had become depleted, if plates had any unwanted bacterial or fungal contamination or showed desiccation. Day 0 is considered as the first day of adulthood and time points for events (death or censorship) were marked almost daily (Figure 2.1). Total number of dead and censored worms and total number of trials for all experiments in chapters 3.0, 4.0 and 5.0 is shown in Appendix 8.1 (Table 8.1a, 8.1b and 8.1c respectively).

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Figure 2.1: Workflow for setting up a lifespan assay, (a) NGM agar plates are made and (b)

seeded with E. coli. (c) Droplets of alkaline hypochlorite solution is spotted onto the plate and adult worms containing eggs are placed in the solution to release the eggs. (d) This generates a synchronised population which can then be (e) transferred onto an experimental (treatment) plate and lifespan can be (f) measured as alive, dead or censored.

Worms were classified into 3 categories at each measured time point: alive, dead or censored. They were classified as alive if any movement is observed. If no movement is observed the agar surrounding the worm is tapped with the sterile worm pick. If still no movement is observed, then the head of the worm is gently tapped with the worm pick to elicit a physical response. If after this there is still no response the worms are classified as dead and are removed from the plate. Worms can also be classified as censored from the experiment if a final time of death is unable to be determined. Censorship of worms occurs if worms do not naturally die from old age. Some examples where censorship would occur are internal hatching of eggs (bagging), bacterial or fungal contamination, vulval protrusion and rupturing or death

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due to worms crawling off of the agar. Although worms are censored from lifespan experiments they are still included for statistical analyses.

2.2.3 C. elegans lifespan assays from digital recording

Flatbed photo scanners were used for some lifespan assays where manual counting assays could not be conducted (for example in hypoxic environments). Use of flatbed scanners to conduct automated lifespan measurements such as with the ‘automated wormscan’ (Mathew et al., 2012) and ‘the C. elegans lifespan machine’ (Stroustrup et al., 2013) inspired the use of flatbed photo scanners for acquisition of photos. The Epson Perfection V800 Photo Scanner was used to scan the agar plates (up to 12 at a time) at least twice daily to generate high resolution images of all plates (Figure 2.2). Once images were acquired the individual plates were analysed manually by comparing images taken. If worms had moved position then they were classified as alive, had they not moved they were classified as dead. Additionally, missing worms (crawled off the plate) were classified as censored along with worms which were seen to have had internal hatching of eggs (bagging), bacterial or fungal contamination, vulval protrusion and rupturing.

Figure 2.2: Digital lifespan recording. (a) Loading plates onto scanners. (b) Representative

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2.2.4 C. elegans treatments

2.2.4.1 Temperature Intervention

In temperature intervention studies, C. elegans (L4 stage) were incubated at an initial temperature (either 15°C or 25°C) for a certain time period of their lifetime and then incubated at the alternative temperature (25°C or 15°C) for the remaining length of their lifetime. C.

elegans N2 and trpa-1(ok999) strains were used in switching experiments. Temperature

switching was conducted by transferring worms between incubators pre-set at either 15C or 25C.

2.2.4.2 Pharmacological Intervention

Pharmacological intervention was used to influence lifespan of C. elegans to identify how drugs affect the lifespan of worms, specific populations and parameters. In these studies, the pharmacological intervention occurred from day 0 (synchronised worms at L4 stage) for the entire adult lifetime, until death.

2.2.4.2.1 Carvacrol

Carvacrol (Figure 2.3; 282197) is a monoterpene phenol found in Oregano and other aromatic plants and is known to have antibacterial properties (Xu et al., 2006). However, it also acts as an activator of human TRPA1 and TRPV3 ion channels. It has previously been shown to allow Ca2+ _{ion entry into cells. Carvacrol (0mM, 50mM, 200mM and 500mM; Solvent: DMSO;}

Control) was added to melted NGM agar before pouring into 55mm plates culture plate.

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2.2.4.3 Hypoxia

C. elegans (L4) were exposed to hypoxia (1% O2, 99% N2) by placing them inside the hypoxia

O2 Control InVitro Glove Box (Coy Laboratories). Temperature was regulated using a climate

control system in the room to an average temperature of 20C. As previously mentioned (section 2.2.3), a flatbed photo scanner (EPSON V800) was used to take images of worms in the hypoxia chamber to generate survival curves.