Examine Chemoavoidance Behavior in Transgenic Worms Using Osmotic Avoidance Assay

Protokol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

NOTE: See Table 1 for the recipes of solutions used in this protocol.

1. Preparation of materials for the Caenorhabditis elegans assay

1. Maintenance of C. elegans strains
   1. Obtain C. elegans (AM141 and HA759) and Escherichia coli (OP50 and NA22) strains (see the Table of Materials).
   2. Maintain the nematodes on the nematode growth media (NGM) plate seeded with E. coli OP50 at 20 °C for AM141 or 15 °C for HA759.
2. Preparation of E. coli OP50 bacterial culture
   1. Pick a single colony of E. coli OP50 from a Luria-Bertani (LB) streak plate and inoculate it into 50 mL of liquid LB culture.
   2. Incubate the OP50 bacteria in a shaker at 37 °C and 200 rpm until an optical density of ~0.5 at 570 nm (OD₅₇₀).
   3. Store the OP50 bacterial culture at 4 °C and use it within two weeks.
3. Preparation of NGM plates with OP50 bacteria
   1. Add 20 g of agar, 2.5 g of peptone, 3.0 g of NaCl, and 975 mL of deionized water to a 1 L autoclavable bottle. Autoclave at 121 °C for 30 min.
   2. Place the liquid NGM agar bottle on the bench to cool to ~60 °C, and then add the following sterile stock solutions: 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 1 mL of 5 mg/mL cholesterol, and 25 mL of 1 M potassium phosphate (pH 6.0).
   3. Pour 20 mL of NGM into a sterile 90 mm Petri dish, and leave the plates on the bench to cool and solidify. Keep the plates upside down and allow them to dry on the bench at room temperature for 2 days.
   4. Dispense 200 µL of the OP50 bacterial culture onto each NGM plate and spread evenly with a sterile glass coating rod. Close the lids and incubate the plates overnight at 37 °C.
   5. Store the NGM plates seeded with OP50 in a plastic box with a cover at room temperature and use within two weeks.
4. Preparation of age-synchronized C. elegans population
   1. Collect gravid adult nematodes into a sterile 1.5 mL microcentrifuge tube and centrifuge at 1000 × g for 1 min. Wash three times and resuspend the nematodes in M9 buffer.
   2. Add an equal volume of bleach solution and agitate gently for 3-5 min. Monitor the bleaching every 15 s under a dissecting microscope.
      NOTE: The bleach solution must be prepared freshly before use by mixing equal volumes of 10% NaOCl and 1 M NaOH (Table 1).
   3. Once most of the nematodes are broken, stop the digestion by diluting with M9 buffer. Quickly centrifuge to remove the supernatant and resuspend the pellet in M9 buffer to repeat the washing three times.
   4. Resuspend the pellet in S medium. Let the nematode residues settle down by gravity for 2-3 min while the eggs remain in the supernatant.
   5. Aspirate the supernatant into a new sterile microcentrifuge tube. Collect the eggs by centrifugation at 1000 × g for 1 min.
   6. Discard ~80% of the supernatant and transfer the eggs into a sterile flask containing 20 mL of S medium. Place the flask in a shaker and incubate the eggs overnight without food at 120 rpm to obtain synchronized L1 nematodes.

2. PolyQ-mediated neurotoxicity assays

1. Treatment of C. elegans with test samples
   1. To prepare nematodes for the polyQ neurotoxicity assay, transfer 300-500 synchronized L1 larvae of HA759 to each well of a 48-well plate with 500 µL of S medium containing OP50 (OD570 of 0.7-0.8) and 5 mg/mL of astragalan, typically three replicate wells for each treatment.
   2. Seal the plate with parafilm and incubate at 15 °C and 120 rpm for 3 days.
   3. Collect the nematodes by centrifugation and wash 3-5 times with M9 buffer. Resuspend the nematodes in M9 buffer for use in neuronal survival and avoidance assays.
2. Osmotic avoidance assay
   1. Divide a food-free NGM plate (9 cm) into normal (N) and trap (T) zones by an 8 M glycerol (~30 µL) line in the middle. Spread a 200 mM sodium azide (~20 µL) line at ~1 cm away from the glycerol line to paralyze the nematodes crossing through the glycerol barrier into Zone T.
   2. Transfer ~200 nematodes each onto Zone N of three replicate plates for each group. Add a drop of 1% butanedione (~2 µL) onto Zone T (1 cm from the plate edge) to attract the nematodes. Cover the lid of the Petri dish immediately, and incubate at 23 °C for 90 min.
   3. Score the number of nematodes on the N and T zones under a microscope. Calculate the avoidance index using eq.
      Avoidance index = N / (T + N)
      Where N and T are the number of nematodes in N and T zones, respectively. Data are presented as means ± standard deviation (SD) of three replicates, representative of >3 independent experiments.
   4. Perform an unpaired, two-tailed t-test to compare the data from the astragalan and control groups.

Table 1:

Solution	Preparation instructions	Storage
1 M CaCl2	111 g CaCl2	Store at room temperature (RT)
	Add dH2O to 1 L
	Autoclave
1 M Potassium phosphate (pH 6.0)	108.3 g KH2PO4	Store at RT
	35.6 K2HPO4
	Add dH2O to 1 L
	Autoclave
1 M MgSO4	246 g MgSO4·7H2O	Store at RT
	Add dH2O to 1 L
	Autoclave
Cholesterol in ethanol (5 mg/mL)	0.5 g Cholesterol	Store at -20 °C
	Add ethanol to 100 mL
	Do not autoclave!
1 M Potassium citrate (pH 6.0)	Add 10.0 g citric acid monohydrate	Store at RT
	Add 146.75 g tri-potassium citric acid monohydrate
	Add dH2O to 1 L
	Autoclave
Trace metals solution	0.93 g Disodium EDTA	Store in dark at RT
	0.345 g FeSO4·7H2O
	0.1 g MnCl2·4H2O
	0.145 g ZnSO4·7H2O
	Add 0.0125 g CuSO4·5H2O
	Add dH2O to 500 mL
	Autoclave
1 M NaOH	4 g NaOH	Store at RT
	Add dH2O to 100 mL
Bleach solution	0.5 mL of 1 M NaOH	Prepare freshly before use
	0.5 mL of 10% NaOCl
M9 buffer	5 g NaCl	Store at RT
	3 g KH2PO4
	6 g Na2HPO4
	Add dH2O to 1 L
	Autoclave
	Add 1 mL of 1 M MgSO4
S basal	5.85 g NaCl	Store at RT
	6.0 g KH2PO4
	1.0 g K2HPO4
	Add dH2O to 973 mL
	Autoclave, and cool to ~60 °C
	Add 1 mL of 5 mg/mL cholesterol in ethanol
S medium	S basal	Store at RT
	Add 3 mL of 1 M CaCl2
	Add 3 mL of 1 M MgSO4
	Add 10 mL of 1 M potassium citrate
	Add 10 mL of trace metals solution
	All components have been autoclaved, do not autoclave
5 mg/mL of astragalan	0.1 g astragalan	Store in aliquots at -80 °C
	Add 20 mL of S medium
	Filter through 0.22 μm syringe filter
200 mM of sodium azide	1.3 g NaN3	Store at 4 °C
	Add 100 mL of S medium
8 M Glycerol	73.67 g glycerol	Store at RT
	Add dH2O to 100 mL
10% Butanedione stock	Mix 10 μL of butanedione with 90 μL of dH2O	Store at RT
1% Butanedione	Add 10 μL of butanedione stock to 90 μL of dH2O	Store at RT
1 L of Luria-Bertani (LB) culture	10 g NaCl	Store at RT
	10 g Tryptone
	5 g Yeast extract
	Add dH2O to 1 L
	Autoclave

Malzemeler

Name	Company	Catalog Number	Comments
C. elegans strains
HA759 rtIs11 [osm-10p::GFP + osm-10p::HtnQ150 + dpy-20(+)]	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/HA759
E. coli strains
NA22	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/NA22
OP50	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/OP50
Reagent
Agar	Shanghai EKEAR Bio-Technology Co., Ltd.	EQ1001-500G	https://www.ekear.com
Butanedione	Sinopharm Chemical Reagent Co., Ltd.	80042427	https://www.reagent.com.cn/goodsDetail/d027c00e64c9404d9aa41391fbb 595d0
Cholesterol	Sigma-Aldrich	C8667	https://www.sigmaaldrich.cn/CN/zh/product/sigma/c8667?context=product
Glycerol	Aladdin Co., Ltd.	G116203	https://www.aladdin-e.com/zh_cn/g116203.html
Peptone	Guangdong HuanKai Microbial Science and Technology Co., Ltd.	050170B	https://www.huankai.com/show/21074.html
Sodium azide	Sinopharm Chemical Reagent Co., Ltd.	80115560	https://www.reagent.com.cn/goodsDetail/5e981aa807664e26af 551e96ff5f07cd
Sodium hydroxide	Sinopharm Chemical Reagent Co., Ltd.	10019718	https://www.reagent.com.cn/goodsDetail/450dfdb1132a4d8a817 d3d8c68ec25e6
Sodium hypochlorite solution	Guangzhou Chemical Reagent Factory	7681-52-9	http://www.chemicalreagent.com/product/DetailProduct.aspx?id=125
Tryptone	Oxoid Ltd.	LP0042B	https://www.thermofisher.cn/order/catalog/product/LP0042B#/LP0042B
Yeast extract	Oxoid Ltd.	LP0021B	https://www.thermofisher.cn/order/catalog/product/LP0021B#/LP0021B
Equipment
48-well cell culture plate	Nest Biotechnology Co., Ltd.	748001	https://www.cell-nest.com/page94?_l=en&product_id=85
90 mm Petri dish	Sangon Biotech (Shanghai) Co., Ltd.	F611003	https://www.sangon.com/productDetail?productInfo.code=F611003
Autoclave	Panasonic	MLS-3781L-PC
Dissecting microscope	ChongQing Optical Co., Ltd.	ZSA0745	http://www.coicuop.com/plus/view.php?aid=64
Microcentrifuge	GeneCompany	GENESPEED X1	https://www.genecompany.com/index.php/Home/Goods/goodsdetails/gid/189.html
Parafilm M	Sigma-Aldrich	P7793-1EA	https://www.sigmaaldrich.cn/CN/en/product/sigma/p7793?context=product
Shaker	Zhicheng Inc.	ZWY-2102C	http://www.zhicheng.net/Product/0865291356.html