Strain selection and maintenance
Caenorhabditis elegans were maintained on nematode growth medium (NGM) agar plates seeded with OP50, which is a slow-growing Escherichia coli mutant. The strains used included N2, eat-2(ad465), isp-1(qm150), clk-1(qm30), daf-1(m40), daf-7(e1372), daf-12(rh286), daf-2(e1370), daf-2(e979), daf-2(e1391), daf-2(e1368), daf-2( e1371), daf-2(m596), daf-2(m577), daf-2(m579), daf-2(Sa193), daf-2(m41), [daf-16(mgDf47); daf-2(e1370)], TJ356(daf-16::GFP), and PD4251(pmyo-3::GFP). All strains were provided by the Caenorhabditis Genetics Center funded by the National Institutes of Health National Center for Research Resources. Unless otherwise stated, all strains were cultured at 15 °C to the L4 stage and then transferred to animals at 20 °C for 2 d.
Freeze–thaw stress conditions
Adult animals (2 d after L4) on NGM plates were washed with M9 buffer and transferred into FACS tube with 1 ml M9 buffer. Caenorhabditis elegans does not survive freezing very well, so to determine optimal freezing length for analyses, we placed animals from room temperature into −80 °C for different amounts of times (4 min, 5 min,6 min, 8 min, 12 min, or 16 min). At 4 min, there was no survival difference between N2 and daf-2(e1370). Ice began to form at 5 min, and all water froze in the tube at 6–7 min; however, all animals, including N2 and daf-2(rf), died at 16 min. At 8 min, there were significant freezing survival differences among mutants. Therefore, animals were treated with freezing stress at −80 °C for 8 min for subsequent analyses. This experiment revealed that freezing stress damaged animals; it is possible that in the body, some cells did not completely freeze, but the freezing stress still injured animals.
Animals were exposed to freezing stress and then thawed at different temperatures (0 °C and 30 °C) for different lengths of time (10 min for 0 °C and approximately 1 min for 30 °C). We removed the tubes at the end of thawing process (when the ice was completely thawed), when the temperature in the tube is still 0 °C.
All treated animals were placed on dry NGM plates for 6 h before determining mortality score with an optical microscope. L1–adult animals were used for the daf-2(rf) survival test for different stages.
Programmed freezing conditions
L1–adult animals on NGM plates were gently washed with M9 buffer. For the freezing procedure, a Cryo 1 °C freezing container was used with a gradient cooling rate of −1 °C/min. Animals were transferred into freezing tubes with 0.9 ml buffer and 0.9 ml 30 % glycerin. At different time points (every 20 or 40 min), animals were thawed in a 30 °C water bath, and the survival rate was then assayed.
Feeding RNAi
dsRNA-expressing E. coli were streaked onto LB agar plates that contained ampicillin (50 μg ml−1) and tetracycline (12.5 μg ml−1), and then incubated at 37 °C overnight. Bacteria were inoculated in 3 ml LB liquid medium that contained only ampicillin (100 μg ml−1) and then incubated at 37 °C overnight. All 3 ml of culture was spun down, the supernatant poured off until 150 μl was left (20× concentrated culture), and pellets were resuspended. Then, 50 μl of cells were resuspended to the center of RNAi plate (NGM/IPTG/ampicillin), allowed to dry (wrapped in aluminum foil), and induced overnight at room temperature (RNAi-seeded plates can be stored at room temperature for 2–3 d before use). Synchronized L1 worms were placed on each plate and incubated at 15 or 20 °C until they reached the desired stage for further experiments.
Scoring mortality
Worms were scored on NGM plates after 6 h of recovery from the thawing procedure. Worms were prodded with a pick at least three times over approximately 10 s; any that failed to move were counted as dead and removed from the plate.
Morphologic cell defects
The PD4251 reporter gene pmyo-3::gfp, which is located in body wall muscle nuclei, was assayed with a fluorescence microscope. pmyo-3::gfp expression of strain PD4251 by RNAi treatment with freezing and thawing was assayed.
Quantitation of locomotion rate by counting body bends
Ten animals were selected from fresh plates with fairly thin lawns, and one worm each was placed new plates. Assays started 24 h later (±1 h). A 3-min timer was used to count number of body bends. Every time the part of the worm just behind the pharynx reached a maximum bend in the opposite direction from the bend last counted was considered one body bend.
daf-16 nucleus translocation
TJ356 nuclear protein daf-16::GFP expression was assayed with a fluorescence microscope. daf-16::GFP expression of [daf-2(RNAi); TJ356] treated with freezing and thawing was assayed compared with that of TJ356.
RNA isolation for qRT-PCR
Total mRNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA) from certain C. elegans and treated with RNase-free DNase (Promega, Madison, WI). Then, reverse transcription (RT) was performed with a TaKaRa RNA PCR kit (Takara, Dalian, China) following the manufacturer’s instructions. The primers used are described in Additional file 5: Table S1 and sequenced in the DNA Sequencing Department of Biosune Systems Biology (Shanghai, China).
mRNA reverse transcription and qRT-PCR
One nanogram in vitro transcribed RNA was added to the RNA sample (500–1000 ng). DEPC H2O was added to the RNA sample to 29.5 μl. Then, 0.5 μl each of 1 μg/μl random hexamer and 1 μg/μl poly dT were added. The mixture was incubated at 65 °C for 10 min, immediately put on ice for 5 min, and let stand at room temperature for 10 min. Then, 18.5 μl pre-mixture, which contained 2.5 μl 10 mM dNTP mix, 10 μl 5× first-strand buffer, 5 μl 0.1 M DTT, and 1 μl RNase OUT, was added. The mixture was then mixed and spun, and placed at room temperature for 2 min. Then, 1 μl Superscript II RT was added and gently mixed. The mixture was then spun down and let stand at room temperature for 10 min, incubated at 42 °C for 50 min, and heat inactivated at 70 °C for 15 min. Then, 1 μl RNase H was added to the solution, which was gently mixed, spun down, and blocked at 37 °C for 30 min.
SYBR Green qRT-PCR was performed on the LightCycler® 480 II System (Roche, Pleasanton, CA) using 5 μl 2× SYBR Green master mix (Roche), 2 μl RNAse free water, 1 μl of forward, 1 μl of reverse primer (10 μM), and 1 μl cDNA per reaction. Primer efficiency was assessed by a dilution series, and a dissociation curve was used to assess primer specificity. qRT-PCR data analysis was performed using genEx software (MultiD).
Statistical analyses
Percent survival was reported as mean ± SEM per trial. Every test was repeated at least three times under the same conditions. Survival rate was analyzed by the nonparametric Mann–Whitney test. Correlation analysis between daf-2(rf) allele freeze–thaw survival and lifespan/other stress traits were conducted by the nonparametric Spearman correlation test.
Availability of supporting data
Primer sequences,data are accessible through this link: https://mynotebook.labarchives.com/share/fangny/MjMuNHwxMzY4MDYvMTgvVHJlZU5vZGUvMzYxNTQ3NzkyNXw1OS40.