1. Observation of SKN-1 Localization in Response to Streptococcus gordonii Infection<ol><li>Add ~100 L4 larvae to each Todd&#8211;Hewitt yeast extract (THY) seeded with Streptococcus and to each nematode growth medium (NGM) agar plate seeded with Escherichia coli (E. coli). Use three plates per strain of bacteria. Incubate the plates for 2 to 3 h at 25 &#176;C.</li><li>Thereafter, remove the plates from the incubator, wash them with a modified M9 buffer (M9W), and collect the worms in 15 mL conical tubes.</li><li>Wash the worms 3x.</li><li>Remove most of the M9W by aspiration and add 500 &#181;L of M9W containing 2 mM sodium azide or 2 mM tetramisole hydrochloride to the worm pellet. This will anesthetize the worms, ensuring that no movement occurs when imaged under the microscope. CAUTION: Use personal protective equipment (PPE) when handling sodium azide. Prepare the azide solution under a chemical hood.</li><li>Incubate the worm pellets at room temperature (RT) for 15 min. Then, spot 15 &#181;L of the worm suspension onto a prepared agarose pad. Gently place a no. 1.5 coverslip over the agarose pad containing the anesthetized worms.</li><li>Using a fluorescent microscope, visualize the localization of Skinhead family transcription factor 1 or SKN-1:GFP (Green Fluorescent Protein-tagged SKN-1) utilizing fluorescein isothiocyanate (FITC) and 4&#8242;,6-diamidino-2-phenylindole (DAPI) filters. Image worms at 10x and 20x magnifications.</li><li>Score the worms based on the level of localization of SKN-1. No nuclear localization, localization of SKN-1B/C::GFP (Green Fluorescent Protein-tagged SKN-1 isoform B/C) in the anterior or posterior of the worm, and nuclear localization of SKN-1B/C::GFP in all intestinal cells are categorized as low, medium, and high levels of localization, respectively.</li><li>After scoring the fluorescent micrographs, determine the statistical differences by chi- squared and Fisher's exact tests using statistical software.</li></ol>

<table class='ck-table-resized'><tbody><tr><td>Media and chemicals</td><td>&#160;</td><td>&#160;</td><td>&#160;</td></tr><tr><td>BD Bacto Todd Hewitt broth</td><td>Fisher Scientific</td><td>DF0492-17-6</td><td>&#160;</td></tr><tr><td>15 mL Conical sterile polypropylene centrifuge tubes</td><td>Fisher Scientific</td><td>12-565-269</td><td>&#160;</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>&#160;</td></tr><tr><td>Tetramisole hydrochloride</td><td>Sigma Aldrich</td><td>L9756</td><td>&#160;</td></tr><tr><td>No.1.5 18 mm x 18 mm cover slips</td><td>Fisher Scientific</td><td>12-541A</td><td>&#160;</td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9539-50G</td><td>&#160;</td></tr><tr><td>LD002</td><td>IdIs1</td><td>SKN-1B/C::GFP + rol-6(su1006)</td><td>Keith Blackwell</td></tr></tbody></table>

visualizing pathogen-induced oxidative stress in c. elegans using gfp-tagged skn-1

Source: <a href='https://app.jove.com/v/59301/studying-oxidative-stress-caused-mitis-group-streptococci'>Naji, A., et al. Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans. J. Vis. Exp. (2019)</a>This video demonstrates the use of green fluorescent protein-tagged SKN-1 in C. elegans to monitor oxidative stress by tracking green fluorescence. Worms exposed to S. gordonii show strong nuclear fluorescence, confirming pathogen-induced stress, while those fed E. coli display diffuse green fluorescence in the cytoplasm, indicating minimal SKN-1 activation.

Visualizing Pathogen-Induced Oxidative Stress in C. elegans Using GFP-Tagged SKN-1

Source: Naji, A., et al. Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans. J. Vis. Exp. (2019)This video ...

https://cloudfront.jove.com/files/ftp_upload/24504/24504_article_thumb_24504.jpg

Begin with two plates containing C. elegans worms that express green fluorescent protein-tagged SKN-1, a transcription factor localized in the intestinal cytoplasm. These worms also contain intestinal lipofuscin granules that show autofluorescence.One plate is seeded with non-pathogenic bacteria as the control group, the other with pathogenic bacteria as the test group.In the test group, hydrogen peroxide produced by the pathogen induces oxidative stress, triggering SKN-1 translocation to intestinal nuclei.In the control group, the majority of SKN-1 remains diffusely distributed in the cytoplasm.Wash each plate with a buffer and transfer the worms into tubes.Centrifuge to separate the worms and remove the buffer.Add a medium containing sodium azide to anesthetize them.Mount the worms on agarose pads, place coverslips, and observe under a fluorescence microscope. Use autofluorescence to visualize the nuclear boundaries.Strong nuclear SKN-1 signal in test, relative to the diffuse cytoplasmic signal in controls, indicates nuclear relocalization and confirms pathogen-induced oxidative stress.Using a pipette, add approximately 100 L4 larvae to each THY streptococcus seeded and NGM E.coli seeded plates. Use three plates per strain of bacteria.Incubate the plates for 2 to 3 hours at 25 degrees Celsius. Thereafter, remove the plates from the incubator. Wash them with M9W and collect the worms in 15 milliliter conical tubes.Wash the worms three times as done previously in the centrifugal step. Remove most of the M9W by aspiration. And add 500 microliters of M9W containing 2 millimolar sodium azide or tetramisole hydrochloride to the worm pellet for anesthesia.Incubate the tube with worm pellets at room temperature for 15 minutes. Then, drop 15 microliters of the worm suspension on to a prepared agarose pad. Under a fluorescent microscope, visualize the localisation of SKN-1 utilizing FITC and DAPI filters.Image worms at 10 times and 20 times magnifications. Score the worms based on three levels of localisation; low localisation, where no nuclear localisation is observed, medium localisation, with SKN-1 BCGFP in the anterior or posterior of the worm, and high localisation, where nuclear localisation of SKN-1 BCGFP is observed in all intestinal cells.

visualizing-pathogen-induced-oxidative-stress-c-elegans-using-gfp

Research

JoVE Encyclopedia of Experiments

Microbiology

Bacterial Pathogenesis and Host Interactions

bacterial-pathogenesis-and-host-interactions

Bacterial Virulence & Biofilms

125 Views. Begin with two plates containing C. elegans worms that express green fluorescent protein-tagged SKN-1, a transcription factor localized in the intestinal cytoplasm. These worms also contain intestinal lipofuscin granules that show autofluorescence.One plate is seeded with non-pathogenic bacteria as the control group, the other with pathogenic bacteria as the test group.In the test group, hydrogen peroxide produced by the pathogen induces oxidative stress, triggering SKN-1...

Visualizing Pathogen-Induced Oxidative Stress in C. elegans Using GFP-Tagged SKN-1

In This Article

Abstract

Protocol

Materials

Reprints and Permissions

Tags

Related Articles

Name	Company	Catalog Number	Comments
Media and chemicals
BD Bacto Todd Hewitt broth	Fisher Scientific	DF0492-17-6
15 mL Conical sterile polypropylene centrifuge tubes	Fisher Scientific	12-565-269
Sodium azide	Sigma Aldrich	S2002-25G
Tetramisole hydrochloride	Sigma Aldrich	L9756
No.1.5 18 mm x 18 mm cover slips	Fisher Scientific	12-541A
Agarose	Sigma Aldrich	A9539-50G
LD002	IdIs1	SKN-1B/C::GFP + rol-6(su1006)	Keith Blackwell