Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: Recommended recipes for reagents used. All the exact recipes of the reagents used in this protocol are outlined here. Specific companies where reagents were purchased are also available in the Table of Materials . Many different sources of chemicals were tested, and those listed in the are those that exhibited the most robust and reproducible results.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Imaging, Control

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: Table of Materials

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Solvent, Imaging, Software

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: Transcriptional reporters for assessing activation of cellular stress responses. The strains listed here are all available through CGC or through special requests to laboratories for use in both qualitative and quantitative imaging methods described in this manuscript. These strains are all derived from the Bristol N2 background. Recommended methods to apply stress to activate the reporters are also provided. All reporters, with the exception of sod-3p::GFP 45 and T24B8.5p::GFP 46 are described in the text.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Activation Assay, Imaging, Derivative Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: Recommended settings for fluorescent microscopy and quantification using a large particle biosorter. This table serves as guideline for recommended exposure times for fluorescent microscopy or PMT values for the large particle biosorter. These will serve as good starting points, but the exposure time and PMT value should be adjusted for every experiment to ensure that no saturation occurs and that fluorescent values are over the detection limit of background signal. If the sample with the brightest signal for an experiment is known (e.g. positive controls for stress induction), those samples can be used to determine the highest exposure time or PMT that can be used without saturating signal. If the brightest samples are not known, then the control can be used and an exposure time or PMT at the center of the dynamic range of your system can be used.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Microscopy, Control

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: (A) Representative fluorescent micrographs of hsp-4p::GFP expressing animals grown on control empty vector (EV) or xbp-1 RNAi. Animals were grown on RNAi from hatch until L4 at 20 °C, then treated with 25 ng/μL tunicamycin or 1% DMSO floating in M9 at 20 °C for 4 hours, and recovered on an OP50 plate for 16 hours at 20 °C prior to imaging. Animals were paralyzed in 100 μM sodium azide on an NGM agar plate and imaged using a Leica M205FA stereomicroscope. (B) Quantitative analysis of (A) using a Union Biometrica large particle biosorter. Data is represented as integrated fluorescence intensity across the entire animal where each dot represents a single animal; DMSO control is in grey and tunicamycin treated animals are in red. Central line represents the median, and whiskers represent the interquartile range. n = 123-291 animals per strain. *** = p < 0.001 using non-parametric Mann-Whitney testing.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Expressing, Control, Plasmid Preparation, Imaging, Fluorescence, MANN-WHITNEY

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: (A) Representative fluorescent micrographs of hsp-6p::GFP expressing animals grown on control empty vector (EV), cco-1, mrps-5, or nuo-4 RNAi. Animals were grown on RNAi from hatch and imaged on day 1 of adulthood at 20°C. Animals were paralyzed in 100 μM sodium azide on an NGM agar plate and imaged using a Revolve ECHO R4 compound microscope. (B) Quantitative analysis of (A) using a Union Biometrica large particle biosorter. Data is represented as integrated fluorescence intensity across the entire animal where each dot represents a single animal; EV control is in grey RNAi-treated animals are in red. Central line represents the median, and whiskers represent the interquartile range. n = 303-384 animals per strain. *** = p < 0.001 compared to EV control using non-parametric Mann-Whitney testing. (C) Representative images of hsp-6p::GFP animals treated with DMSO or Antimycin A. Animals were grown from hatch on 0.2% DMSO plates and transferred to plates containing 0.2% DMSO or 3 mM antimycin A for 16 hours prior to imaging on a Revolve ECHO R4 compound microscope. All growth was performed at 20°C. (D) Quantitative analysis of (C) using a large particle biosorter similar to (B). DMSO controls are in great, and Antimycin A-treated animals are in red. n = 495 for DMSO and 219 for Antimycin A. *** = p < 0.001 compared to EV control using non-parametric Mann-Whitney testing.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Expressing, Control, Plasmid Preparation, Microscopy, Fluorescence, MANN-WHITNEY, Imaging

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: (A) Representative fluorescent micrographs of gst-4p::GFP expressing animals grown on control empty vector (EV), skn-1, or wdr-23 RNAi. Animals were grown on RNAi from hatch until L4 stage at 20 °C. Animals were grown on RNAi from hatch until L4 at 20 °C, then treated with 2 mM TBHP in M9 or only M9 for “untreated” control at 20 °C for 4 hours, and recovered on an EV plate for 16 hours at 20 °C prior to imaging. Animals were paralyzed in 100 μM sodium azide on an NGM agar plate and imaged using a Revolve ECHO R4 compound microscope. (B) Quantitative analysis of (A) using a Union Biometrica large particle biosorter. Data is represented as integrated fluorescence intensity across the entire animal where each dot represents a single animal; untreated control is in grey and TBHP-treated animals are in red. Central line represents the median, and whiskers represent the interquartile range. n = 101-204 animals per strain. *** = p < 0.001 compared to respective EV control using non-parametric Mann-Whitney testing.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Expressing, Control, Plasmid Preparation, Imaging, Microscopy, Fluorescence, MANN-WHITNEY

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: (A) Representative fluorescent micrographs of hsp16.2p::GFP expressing animals grown on control empty vector (EV) or hsf-1 RNAi. Animals were grown on RNAi from hatch at 20 °C until day 1. Day 1 animals were either left at 20 °C (untreated) or exposed to 2 hours of heat stress at 34 °C, then recovered for 2 hours at 20 °C. Animals were paralyzed in 100 μM sodium azide on an NGM agar plate and imaged using a Leica M205FA stereomicroscope. (B) Quantitative analysis of (A) using a Union Biometrica large particle biosorter. Data is represented as integrated fluorescence intensity across the entire animal where each dot represents a single animal; untreated control is in grey and heat-shocked animals are in red. Central line represents the median, and whiskers represent the interquartile range. n = 320-364 animals per strain. *** = p < 0.001 using non-parametric Mann-Whitney testing. (C) Representative fluorescent micrographs of hsp-70p::GFP expressing animals grown on control EV and hsf-1 RNAi and treated as described in (A). (D) Quantitative analysis of (C) as described in (B). n = 773-941 animals per strain.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Expressing, Control, Plasmid Preparation, Fluorescence, MANN-WHITNEY

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: (A) Lifespans of nematodes grown on 1% DMSO containing 25 ng/μL tunicamycin (TM) plates. Animals were grown on 1% DMSO plates from hatch until day 1, and transferred to respective TM plates at day 1. Animals were kept on control empty vector (EV) or xbp-1 RNAi from hatch until the end of the assay at 20 °C. Adult animals are manually moved away from progeny every day until ~ day 7-8 when progeny were no longer detected, then scored every 2 days until all animals were recorded as dead or censored. Animals with bagging, vulval protrusions/explosions, or those that crawled up the sides of plates were considered censored. (B) Survival curve of nematodes in 100 mM paraquat (PQ) dissolved in M9 solution. Animals were grown on EV or daf-2 RNAi from hatch until day 1 of adulthood at 20 °C. Animals were placed into 50 μL of M9 + PQ solution in a 96 well-plate at 20 °C and visualized every 2 hours until all animals were motionless. (C) Survival curve of nematodes at 37 °C. Wild-type (N2), ttx-3(KS5), and sur-5p::hsf-1 animals were grown on EV plates from hatch until day 1 at 20 °C. At day 1, animals were moved to 37 °C and scored every 2 hours until all animals were scored as dead or censored. (D) Pooled data of all thermotolerance assays performed at 37 °C. Data are represented as percent alive at hour 9 of a thermotolerance assay, with each line representing a matched experiment performed on the same day. (E) Pooled data of all thermotolerance assays performed at 34 °C. Data are represented as percent alive at hour 14 of a thermotolerance assay, with each line representing a matched experiment performed on the same day. All statistics for A-C were performed using Log-Rank (Mantel-Cox) testing and can be found in Table 4.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Control, Plasmid Preparation

Journal: Journal of visualized experiments : JoVE

Article Title: Measurements of physiological stress responses in C. elegans .

doi: 10.3791/61001

Figure Lengend Snippet: Statistics for lifespans and stress survival assays. All sample sizes, statistics, and censorship rates for are available here.

Article Snippet: NGM RNAi plates , 1 mM CaCl2, 5 μ/mL cholesterol, 25 mM KPO4 pH 6.0, 1 mM MgSO4, 2% (w/v) agar, 0.25% (w/v) Bacto-Peptone, 51.3 mM NaCl, 1 mM IPTG, 100 μ/mL carbenicillin/ampicillin. Store at 4 ° C in dark for up to 3 months.

Techniques: Plasmid Preparation