Worm culture and imaging

Abstract

This study explores C. elegans as a model to understand wound healing, gene editing using CRISPR-Cas9, and dynamic biological changes via live imaging. Detailed protocols are provided for culturing worms, performing CRISPR-Cas9 knock-in, generating transgenic lines, conducting laser wounding assays, and analyzing fluorescent signals in live worms.

Keywords

C. elegans, CRISPR-Cas9, Laser Wounding, Live Imaging, Gene Editing, GFP Puncta Quantification, Statistical Analysis

Materials and Reagents

• C. elegans strains: N2 Bristol (WT strain), C42D4.3–8xMS2 (syb5468), mai-1–8xMS2 (syb5458)

• NGM (Nematode Growth Medium) plates with E. coli OP50

• Levamisole (12 µM) (Sigma-Aldrich®)

• Bleaching reagents (Sodium hypochlorite solution 20%) 

• Plasmids: Psemo-1-MCP-24xSuntag, Pcol-19-antibody-sfGFP, Pttx-3-RFP

• ClonExpress Mulit Cloning Kit (Vazyme)

Equipment and software

• Standard microinjection setup

• Spinning disk confocal microscope Nikon Eclipse Ti with Andor confocal scanning unit, The confocal should provide magnification up to 100x for better visualization. 

• Fiji software for image analysis and foci measurement (https://imagej.net/imagej-wiki-static/Fiji)

• GraphPad Prism for statistical analysis

Procedure

1. Worm Culture

1. Prepare NGM Plates:

   • Melt agar with NaCl, K₂HPO4, KH₂PO₄, KOH and Peptone, add Cholesterol (1 mg/mL), CaCl₂ (1 mM), MgSO₄ (1 mM), and buffer.

   • Pour 12 mL per petri dish and allow to solidify.

   • Seed plates with E. coli OP50, incubate overnight at 37°C.

2. Worm Culturing Conditions:

   • Maintain worms at 20–22.5°C on seeded NGM plates.

   • Transfer worms every 2–3 days to fresh plates to avoid starvation.

3. Synchronization:

   • Bleach gravid adults with hypochlorite solution to isolate embryos.

   • Incubate in M9 buffer overnight for L1 synchronization.

   • Transfer synchronized larvae to NGM plates and incubate until Day 1 (L4 + 24 hours).

2. CRISPR-Cas9 Knock-In

1. Design sgRNA and Repair Templates:

   • Identify target genes (C42D4.3 and mai-1) and insert MS2 sequences at the downstream of the stop codon.

   • Use Gibson Assembly to clone the repair templates.

2. Microinjection:

   • Mix sgRNA, repair template, and Cas9-NLS-pU6-dpy-10 sgRNA in injection buffer.

   • Add Pmyo-2-mCherry as a co-injection marker.

   • Inject into the gonads of  Day 1 adults.

3. Screening and Validation:

   • Screen for Roller/Dumpy worms under a fluorescence microscope.

   • Perform PCR genotyping and sequencing to confirm knock-in events.

3. Transgenic Worm Generation

1. Injection of Plasmids:

   • Mix Pcol-19-BFP-8xMS2 (10 ng/µL), Psemo-1-MCP-24xSuntag (50 ng/µL), Pcol-19-antibody-sfGFP (10 ng/µL), and Pttx-3-RFP (10 ng/µL).

   • Inject plasmid mixture into synchronized Day 1 adults.

2. Strain Generation:

   • Screen for transgenic worms with visible fluorescent markers.

   • Maintain strains by transferring fluorescent individuals to fresh plates.

4. Laser Wounding Assay

1. Sample Preparation:

   • Mount Day 1 adult worms on 4% agarose pads on glass slides.

   • Anesthetize with 12 µM Levamisole to immobilize worms.

2. Laser Parameters:

   • Use a Micropoint UV laser system with energy set to 65–70.

   • Set repetition rate to 10 Hz, firing five times per wounding.

3. Wounding Locations:

   • Target anterior and posterior one-third sections of the worm body.

5. Live Imaging

1. Microscopy Setup:

   • Use a spinning disk confocal microscope (×100, NA 1.46).

   • Set time-lapse imaging to capture every 2 seconds for 930 seconds (30 seconds pre-wounding, 900 seconds post-wounding).

2. Imaging Parameters:

   • GFP (488 nm laser, 280 ms exposure)

   • RFP (561 nm laser, 120 ms exposure)

6. Quantification of GFP Puncta

1. Preparation of Image Data

1. Open Images in Fiji/ImageJ:

   • Import confocal images into Fiji/ImageJ software.

   • Ensure that all relevant channels (e.g., GFP for green fluorescence, RFP for red fluorescence) are properly loaded.

2. Set the Region of Interest (ROI):

   • Identify the wound site in the images.

   • Select an 600 × 400-pixel region around the wound area for analysis to standardize measurements across samples.

2. Background Subtraction and Thresholding

1. Background Subtraction:

   • Use the "Subtract Background" function in ImageJ to remove uneven illumination.

   • Set the rolling ball radius according to the fluorescence signal intensity to optimize background removal.

2. Set Threshold:

   • Apply the "Threshold" function to convert the image into a binary format (black and white).

   • Use the background intensity as the threshold value. This can be done by selecting "Adjust > Threshold" and manually adjusting to exclude non-specific signals while retaining puncta/clusters.

3. Quantification of Clusters

1. Analyze Particles:

   • Select "Analyze > Analyze Particles" to quantify the size, intensity, and number of clusters in the selected ROI.

   • Set the following parameters in the "Analyze Particles" menu:

     • Size: Minimum size of clusters set to 0.2 square micrometers. Leave the maximum size blank (to include all visible clusters).

     • Circularity: Leave blank unless specific shapes are required.

     • Enable the following options:

       • "Display Results"

       • "Summarize"

       • "Add to Manager" (to save ROIs for reference).

   • Run the analysis to automatically calculate:

     • Size: Total area of each cluster.

     • Number: Total number of clusters within the ROI.

4. Data Export

1. Export Results:

   • Save the "Results" and "Summary" tables generated by the "Analyze Particles" function as .csv files.

   • Export images of the analyzed ROIs and binary outputs for quality control.

2. Statistical Analysis:

   • Import data (size, number) into GraphPad Prism or a similar statistical software for further analysis.

   • Calculate mean and Standard deviation (SD) for each parameter (size and number).

5. Sample Size and Replicates

1. Sample Size:

   • Analyze at least 20 wounded young adult animals to ensure statistical robustness.

2. Reporting Results:

   • Quantify and report size and number changes using mean ± SD.

   • Clearly specify the sample size (n ≥ 20) in the results.