Materials and Reagents

Microscope slides (VWR, catalog number: 48382-171 )
Coverslips for microscope slides (Fisher Scientific, Fisherbrand, catalog number: 12-541A )
60 mm plates (Fisher Scientific, Fisherbrand, catalog number: FB0875713A )
100 mm plates (Fisher Scientific, Fisherbrand, catalog number: FB0875713 )
Labeling tape (Fisher Scientific, Fisherbrand, catalog number: 15-901-5K )
Glass pipette
Aluminum foil
Autoclave tape
Inoculating loops
Pipette tips
C. elegans experimental strain:
RT258: unc-119(ed3); pwIs50[lmp-1::GFP, unc-119]
Note: LMP-1 is the orthologue of mammalian Lamp1 that localizes to lysosomes in Caenorhabditis elegans (Kostich et al., 2000)
C. elegans control strain: N2
Calcium chloride dihydrate (CaCl₂·2H₂O) (Fisher Scientific, catalog number: C79-500 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Fisher Scientific, catalog number: M63-500 )
Potassium phosphate monobasic (KH₂PO₄) (Fisher Scientific, catalog number P285-500 )
Cholesterol (Sigma-Aldrich, catalog number: C8667 )
EtOH (Merck, catalog number: EX0276-4 )
LE agarose for making 2.2% agarose (BioExpress, GeneMate, catalog number: E-3120 )
Levamisole (Sigma-Aldrich, catalog number: 31742 )
C. elegans culture
1. Platinum wire-pick to transfer C. elegans
2. Making NGM plates (Brenner, 1974; He, 2011) (see Recipes)
  Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
  Bacto peptone (BD, Bacto^TM, catalog number: 211677 )
  Bacto agar (BD, Bacto^TM, catalog number: 214030 )
  Double distilled water
  Cholesterol 5 mg/ml in 95% EtOH (see Recipes)
  1 M CaCl₂ sterile (see Recipes)
  1 M MgSO₄ sterile (see Recipes)
  1 M KH₂PO₄ pH 6.0 sterile (see Recipes)
3. Making 2x YT + OP50 for spotting NGM plates
  OP50 frozen stock
  2x YT agar plate
4. 2x YT agar plate (see Recipes)
  Bacto tryptone (BD, Bacto^TM, catalog number: 211705 )
  Yeast extract (Fisher Scientific, catalog number: BP1422-500 )
  Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
  Bacto agar (BD, catalog number: 214030 )
  Double distilled water
5. 2x YT medium (see Recipes)
6. 2x YT medium + OP50 (see Recipes)
2.2% agarose pad (see Recipes)
9 mM levamisole/1x PBS (see Recipes)

Equipment

20 °C Incubator for C. elegans storage (VWR, manufactured by Sheldon Manufacturing, model: Model 2020 )
4 L flask
Stir bar
Stir plate
Autoclave
2 L beaker
1 L bottle
37 °C Incubator (VWR, manufactured by Sheldon Manufacturing, model: 5025 T )
Microwave
Heating block
5-100 ml bottle
Microscope (Carl Zeiss, model: STEMI SV 6 )
Zeiss LSM 510 Meta confocal microscope (Zeiss, model: LSM 510 ):
1. 63x lens
2. Argon 488-nm laser
3. Helium neon 543-nm laser

Software

MetaMorph^® Microscopy Automation & Image Analysis Software (Sunnyvale, CA)

Procedure

Note: Before starting the protocol: Ensure that 60 mm NGM plates (spotted with OP50) have been prepared.
Day 1

Set up three plates with three adult hermaphrodites (one day post-L4 stage) on each spotted 60 mm NGM plate for each C. elegans experimental and control strain.
Leave plates in a 20 °C incubator for three days for the hermaphrodites to lay eggs.

Day 4
Move 50-100 L4 stage C. elegans of each experimental strain onto new OP50-spotted 60 mm NGM plates (the offspring of the P0 hermaphrodites). This ensures that these animals would be fairly synchronized young adults for imaging on the following day.

Day 5: Preparation for microscopy

Prepare the imaging slide by first making the agarose pad. See steps 1-4 in Figure 1 for instructions.
Next, add 7 μl of 9 mM levamisole/1x PBS (see Recipes) to the center of the agarose pad (Figure 1, S5).
Pick 20-30 adult worms of one C. elegans experimental strain and submerge them in the 9 mM levamisole/1x PBS (Figure 1, S5).
Put a cover slip on top of the agarose pad, ensuring that it fully covers the C. elegans and the 9 mM levamisole/1x PBS (Figure 1, S6).
Repeat Steps 1-5 for all experimental and control strains used.
Do microscopy to image LMP-1::GFP (or marker of interest) and focus on the intestine right below the pharynx, using the 63x objective. Take images that show the desired signal while avoiding saturation of the signal.
Image C. elegans wild type strain RT258 first with both the Argon 488-nm laser and the Helium neon 543-nm laser. RT258 should be imaged first because it is the wild type strain which measurements from other experimental strains should be compared against.
Take images of the intestines of approximately 5-10 adult C. elegans for the wild type RT258 strain.
Repeat Steps 7 and 8 for each control and experimental strain. Make sure that images of all strains are taken using the same exposure and magnification as RT258.

Figure 1. Preparation of imaging slides

Data analysis

MetaMorph version 7.5.0.0 was used to analyze confocal microscopy images, though other software could also be used.
All confocal microscopy images were converted to ‘.tif’ format for use with MetaMorph.
Identify the compartments that ONLY have green fluorescence (excited by the 488-nm laser). These compartments are LMP-1::GFP-positive that marks lysosomes. Compartments that are yellow (fluoresce with both 488-nm and 543-nm lasers) are gut granules, which will also be seen in C. elegans control strain N2: these gut granule compartments should not be measured or included for determining sizes of bona fide LMP-1::GFP compartments (Figure 2). We do not know whether the LMP-1::GFP signal decreases in older adults; if it does, this protocol may not be effective at differentiating bona fide GFP signal from autofluorescence.

Figure 2. Representative images of intestinal images. Compartments that fluoresce with both 543-nm and 488-nm excitation are granules while compartments that fluoresce with only 488-nm excitation are LMP-1::GFP-positive compartments. Arrows indicate examples of LMP-1::GFP-positive compartments. Arrowheads indicate examples of gut granules. Yellow circles indicate clumped compartments. Scale bar = 10 μm.
For each LMP-1::GFP positive compartment, use MetaMorph to measure the intensity and area of the selected compartment. See Figure 3 for a pictorial guide (Figure 3).
1. Go to Measure in the tool bar and select ‘Show Region Statistics…’
2. Use the trace region tool to select the LMP-1::GFP positive compartment.
3. Double click on the selected region.
4. To get measurements of LMP-1::GFP-positive compartments’ intensities, use the ‘Average’ Gray Level value. This value is already normalized by the area size.
5. To get measurements of LMP-1::GFP-positive compartment size, use the ‘Area’ value.
  
  Figure 3. Using MetaMorph to measure LMP-1::GFP-positive compartment size and marker intensity
6. Collect measurements of all the LMP-1::GFP-positive compartments for each adult C. elegans intestine, for each strain, from each image. Do not include clumped compartments because it is difficult to get accurate quantitations (due to ambiguity in the location of each individual compartment). The result is approximately 10-20 clearly defined compartments per image.
7. Average all the LMP-1::GFP-positive intensity measurements using Excel to obtain the average LMP-1::GFP-positive intensity for each strain. Average all of the LMP-1::GFP positive size measurements to obtain the average compartment size for each strain. Figure 4 depicts results obtained when comparing the wild type strain with a cup-5(null) strain (CUP-5 is a cation channel; loss of CUP-5 protein results in lysosomal dysfunction) (Fares and Greenwald, 2001b; Hersh et al., 2002). Loss of CUP-5 resulted in an increase in LMP-1::GFP-positive intensity, but no significant change in lysosomal size.

Note: We have successfully used this protocol on strains carrying various mutations or following RNAi treatment.

Figure 4. Representative images of LMP-1::GFP intensity and compartment size data obtained. * indicates P < 0.05.

Notes

The higher the ‘Average’ Gray Level, the larger the lysosomal compartment size.
If imaging multiple strains with the same marker, take all images using identical confocal settings, preferably during the same microscopy session. This would allow comparisons of compartment sizes and intensities between strains/genotypes. Additionally, though not always technically possible, when feasible, strains to be compared should be imaged on the same slide to reduce slide-to-slide variation during microscopy.
Repeat this experiment multiple times to determine reproducibility and statistical significance.
This method can be used to analyze markers for other organelles than lysosomes in adult intestinal cells, distinguishing marker signals from gut granule autofluorescence.

Recipes

Nematode Growth Medium (NGM) agar (3 L)
1. Add the following to a 4-L flask:
  NaCl (9.0 g)
  Bacto peptone (7.5 g)
  Bacto agar (51 g)
  Double distilled water (2,925 ml)
2. Stir bar
3. Autoclave for 1 h
4. Place flask on stir plate, set on low stir, and let cool to 55 °C
5. Once cool, add the following:
  CaCl₂ 1 M sterile (3 ml)
  MgSO₄ 1 M sterile (3 ml)
  KH₂PO₄ 1 M pH 6.0 sterile (75 ml)
  Cholesterol 5 mg/ml in 95% EtOH (3 ml)
6. Pour 8 ml into each 60 mm plate
7. Let dry for one night before storing unspotted plates at 4 °C or spotting with 2x YT + OP50
2x YT agar (1 L)
1. Add the following into a 2-L (or larger) flask:
  NaCl (5.0 g)
  Bacto tryptone (16 g)
  Yeast extract (10 g)
  Bacto agar (15.0 g)
  Double distilled water (Up to 1 L)
2. Stir bar
3. Autoclave for 1 h
4. Place flask on stir plate, set on low stir, and let cool 55 °C
5. Pour 18 ml into each 100 mm plate
6. Wait two days for plates to dry and then store at 4 °C
2x YT medium (1 L)
1. Add the following to a 2-L beaker
  Tryptone (16 g)
  Yeast extract (10 g)
  NaCl (5 g)
  Fill to 1 L with double distilled water
2. Add a stir bar into the beaker and let the mixture stir on a stir plate until homogenous
3. Pour into 1 L bottles
4. Autoclave for 30 min
5. Store at room temperature
2x YT medium + OP50 (1 L)
1. Day 1: Use a pipette tip to spread OP50 frozen stock across a 2x YT agar plate. Place plate in a 37 °C incubator and leave it overnight
2. Day 2: Use an inoculating loop to pick one colony from the 2x YT agar plate and transplant the colony to a 1 L bottle of 2x YT Medium. Place the 1 L bottle into the 37 °C incubator and leave it overnight
3. Day 3: Remove bottle from the 37 °C incubator and store at 4 °C until use
2.2% agarose pad
1. Add the following to a flask:
  Agarose (0.22 g)
  Double distilled water (10 ml)
2. Microwave flask for 60 sec. If the agarose and double distilled water solution is not homogenous, microwave for another 30 sec
3. Store in a tube that is in a heating block (68 °C)
9 mM levamisole/1x PBS
1. Mix the following in a tube:
  9 ml of 10 mM levamisole (Sigma-Aldrich)
  1 ml of 10x PBS
2. Store at room temperature
Cholesterol (5 mg/ml)
1. Add the following to a 500 ml beaker
  Cholesterol (2.5 g)
  100% EtOH (475 ml)
  Double distilled water (25 ml)
2. Add a stir bar in the beaker and let the mixture stir on a stir plate until homogenous
3. Pour 100 ml into five 100 ml bottle (please confirm this description)
1 M CaCl₂
1. In a beaker, dissolve CaCl₂ (73.5 g) into 500 ml double distilled water
2. Add a Stir bar into the beaker and let the mixture stir on a stir plate until homogenous
3. Pour into a 500 ml bottle
4. Autoclave for 30 min
1 M MgSO₄
1. In a beaker, dissolve MgSO₄ (123.25 g) into 500 ml double distilled water
2. Add a Stir bar into the beaker and let the mixture stir on a stir plate until homogenous
3. Pour into a 500 ml bottle
4. Autoclave for 30 min
1 M KH₂PO₄ pH 6.0
1. In a beaker, dissolve KH₂PO₄ (68.05 g) into 450 ml with double distilled water
2. Add a Stir bar into the beaker and let the mixture stir on a stir plate until homogenous
3. Adjust pH to 6.0 with NaOH
4. Fill up to 500 ml with double distilled water
5. Pour into a 500 ml bottle
6. Autoclave for 30 min

Acknowledgments

This protocol was adapted from Huynh et al. (2016). This work was supported by a Microscopy Society of America grant (to J.M.H) and by National Science Foundation grant 3004290 (to H.F.). The authors have no conflicts of interest or competing interests.

References

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