P REGUNTAS DE R ESPUESTA O RAL EN P LENO

15.1.1 Worm handling

To maintain worm populations either a part of a plate containing a growing or starved population was “chunked” with a sterilized spatula to a fresh NGM seeded with OP50. Or single worms of a growing population were transferred with a worm pick (platinum wire) to a fresh NGM seeded with OP50.

15.1.2 Bacterial handling

Bacteria (E. coli) from glycerol stock were streaked to petri dishes with soild LB medium containing corresponding antibiotics. Plates were incubated overnight at 37 °C and the next day a single colony was picked to incubate liquid LB containing corresponding antibiotics. Plates were kept at 4 °C and reused to pick single colonies for no longer than a month. Liquid culture was allowed to grow overnight but at least 16 h, to reach stationary growth phase. 400 µl, 200 µl or 100 µl of liquid overnight culture was then used to seed 10 cm, 6 cm and 3 cm plates.

15.1.3 Cleaning and synchronization Populations with bleaching solution

To clean contaminated plates depending on the population´s stage worms were either washed directly of the plate with M9 or a small chunk was transferred to a new plate. They were incubated than at appropriate temperature until enough animals had reached adulthood and eggs were visible in the germline and then washed of the plate with M9. For synchronization an uncontaminated plate with Worms were allowed to either settle in the tube (1, 5 eppendorf or 15 ml falcon tube) or spinned down for 2 min at 2000 rpm. Supernatant was removed and M9 and bleaching solution (2:3) was added worms were vortexed at RT for around 8 min until only eggs visible under the dissecting microscope. Eggs were then washed 3 times in M9 (2 min, 2000 rpm) and transferred to fresh plates.

To achieve a higher degree of synchronization day two adults of a synchronized population were placed on fresh plates an allowed to lay eggs for two hours at 20 °C.

15.1.5 Dauer assay (hydrophobic compounds)

3.5 cm Petri dishes containing 3 ml nematode growth medium (NGM) where incubated with 50 µl E.coli OP50 in LB (saturated) and incubated over night at RT. 12 µl of 10 mM hydrophobic compound solved in ethanol (EtOH) was added to the surface to a final concentration of 40 µM. EtOH was allowed to evaporate for 1 h before eggs were transferred or pipetted to plates. A synchronized adult daf-2(e1368) population was bleached to receive eggs and approximately 70 eggs were pipetted to prepared plates. After 60 h and 48 h incubation at 22.5°C and 25 °C, respectively the fraction of animals that formed dauer larvae was scored with a microscope.

For dauer assays on RNAi parental generation was grown from egg on, on E. coli HT115 bacteria expressing according RNAi. All clones were picked from Ahringer library , only RNAi clones E02C12.6, C02A12.1, ZK455.4, Y39B6A.13 and Y39B6A.20 were picked from Vidallibrary (Kamath, 2003; Rual et al., 2004).

15.1.6 Dauer assay (hydrophilic compounds)

Powders of hydrophilic compound were weighed out and disolved in liquid NGM (60°C) to a final concentration of 10 mM or 100 mM. 3.5 cm NGM compound Petri dishes (3 ml) where incubated with 50 µl E. coli strain OP50 in LB (saturated) and incubated over night room temperature (RT). One batch was radiated with UV light (6,000 J/cm2

) to prevent further division of the bacteria. A synchronized adult daf-2(e1368) population was treated by bleaching to synchronize eggs. Around 70 eggs were pipetted to prepared plates. After 60 h incubation at 22.5°C the fraction of animals that formed dauer larvae was scored.

15.1.7 Gonadal migration defect

3.5 cm Petri dishes containing 3 ml nematode growth medium (NGM) where incubated with 50 µl E.coli OP50 in LB (saturated) and incubated over night at RT. 12 µl of 10 mM hydrophobic compound solved in ethanol (EtOH) was added to the surface to a final concentration of 40 µM. EtOH was allowed to evaporate for 1 h before eggs were transferred or pipetted to plates. A synchronized adult daf-2(e1368) population was bleached to receive

eggs and approximately 70 eggs were pipetted to prepared plates. After 60 h incubation at 26°C the fraction of animals that had gonadal migration defect was scored with a microscope.

15.1.8 Statiscical Analyzes of dauer and mig phenotype

At least three technical replicates per sample were scored to create one of at least three biological replicate. Fractions of three technical replicates were averaged and at least three biological replicates were used to test for significance with Prism GraphPad.

15.1.9 DAF-9 hypodermal expression

3.5 cm Petri dishes containing 3 ml nematode growth medium (NGM) where incubated with 50 µl E.coli OP50 in LB (saturated) and incubated over night at RT. 12 µl of 2.5 mM hydrophobic compound or compound mix solved in ethanol (EtOH) was added to the surface to a final concentration of 10 µM. For DA a final concentration of 100 nM was used. Worms were synchronized by bleaching and around 2000 eggs were placed on each plate and incubated at 20 °C. L3 animals were then staged and either analyzed via Microscopy () or with the COPA Biosorter.

15.1.10 Lifespan analyzes

In brief, synchronized populations of desired strains were grown reproductively at 20°C for at least two generations. Adults were either allowed to lay eggs on experimental conditions or eggs were kept on NGM till L4 larval stage and then transferred to experimental conditions. Worms were transferred every 24 h during the egg laying period and every other day thereafter. Worms that did not move after being tipped gently with a worm pick were considered as dead and removed from the plate. All lifespan assays were performed at 20°C. All animals that crawl off the plate, showed phenotype of internally hatched larvae (bagged) or rupture of vulva (exploded) were censored and excluded from experiment.

For lifespan assay on UV killed bacteria plates were prepared as described above. In parallel regular 10 cm NGM plates were seeded with OP50 grown over night and subsequently UV treated (12,000 J/cm2

). Dead bacteria were washed off of 10 cm plates with M9 and transferred to experimental plates. To exclude contamination with bacteria coming from worms gut. Worms were bleached and grown to L4 larvae on plates with UV killed bacteria before being transferred to experimental plates.

For lifespan assays on RNAi parental generation was grown from egg on, on E. coli HT115 bacteria expressing according RNAi. All clone were picked from Ahringer library (Kamath, 2003), only RNAi clones E02C12.6, C02A12.1, ZK455.4, Y39B6A.13 and Y39B6A.20 were picked from Vidal library (Rual et al., 2004). Statistical analyses were performed using the log-rank (Mantel-Cox) method with GraphPad Prism software.

15.1.11 Brood size assay

Populations were synchronized by bleaching and incubated at 20 °C till day two of adulthood to make sure parental generation of experimental generation arose from eggs laid by animals at the same age. Parental generation was allowed to lay eggs for 2 h on fresh NGM plates seeded with OP50. After 48 hours at 20 °C L4 animals were singled to at least 20 x 6 cm NGM plates containing either 10 µM 7-KC or appropriate volume EtOH. After 24 h at 20 °C each animals was transferred to a fresh plate and the old plate was incubated another 24 hours before it was either scored with dissecting microscope or stored at 4 °C till scoring (but never longer than 3 days). This procedure was repeated for ten days or till no living eggs were laid. Only eggs that developed at least to L1 larvae were scored as living progeny.

15.1.12 Pharyngeal pumping rate

A population reproductively growing for at least 3 generations was synchronized by 2 h egg lay. After 48 h at 20 °C around 100 L4 worms were transferred to experimental plates containing either 10 µM 7-KC or appropriate volume EtOH. After 24 h (day 1) the number of pumps of the posterior pharyngeal bulb per 20 s was scored under dissecting microscope and multiplied by 3 to determine pumps/min. Animals were transferred every 24 hours during egg lay period and every other day thereafter. Pharyngeal pumping was scored on day 1, day 6 and day 10.