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Plate Assay to Determine Caenorhabditis elegans Response to Water Soluble and Volatile Chemicals
平板分析法检测秀丽隐杆线虫对水溶性和挥发性化学物质的反应   

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Frontiers in Behavioral Neuroscience
May 2017

Abstract

The nematode Caenorhabditis elegans is widely used for behavioral studies ranging from simple chemosensation to associative learning and memory. It is vital for such studies to determine optimal concentrations of attractive and aversive chemicals that C. elegans can sense. Here we describe a resource localization assay in which a chemical compound of interest is placed in two compartments of a quadrant plate in order to determine optimal concentrations of the chemical in behavioral studies. Using the assay, we determined the optimal concentration of a water-soluble attractant, KCl, as an unconditioned stimulus for the study of associative learning and memory. In this protocol, we also describe a chemotaxis assay using a square agar plate spotted with an aversive olfactory cue, 1-nonanol, as a conditioned stimulus.

Keywords: Attractant (引诱), Aversive stimulus (厌恶刺激), Chemosensory behavior (化学感受行为), Learning and memory (学习和记忆), Plate assay (平板分析法)

Background

The nematode Caenorhabditis elegans has extensively been used as a model organism for the study of animal behaviors. C. elegans senses a variety of water-soluble and volatile chemicals that are mainly mediated by amphids, the largest chemosensory organs (Ward, 1973; Dusenbery, 1974; Bargmann and Horvitz, 1991; Bargmann et al., 1993). It is essential for the behavioral study to determine precise concentrations of chemicals that can be sensed by C. elegans. To determine optimal concentrations of water-soluble attractants for C. elegans, Wicks et al. (2000) used a quadrant agar plate for the behavioral assay in which a chemical of interest was mixed with agar in two compartments and this assay has widely been used for many chemicals (e.g., Jansen et al., 2002; Ortiz et al., 2009; Murayama and Maruyama, 2013; Sassa et al., 2013). Chemotaxis assay has also been used to measure the sensitivity of C. elegans to volatile compounds spotted on an agar plate (Bargmann et al., 1993; Troemel et al., 1997). C. elegans is also an excellent model organism for the study of associative learning and memory, in which water-soluble chemicals and volatile chemicals were used as an unconditioned stimulus (US) and a conditioned stimulus (CS) (Amano and Maruyama, 2011; Nishijima and Maruyama, 2017). For effective conditioning of worms, concentrations of CS and US are crucial parameters. The resource localization assay with quadrant agar plates and a chemotaxis assay on square agar plates were successfully used to define optimal concentrations of US and CS for the study of learning and memory. Therefore, these assays could be applied for many other attractive and repulsive chemicals in C. elegans behavioral analysis.

Materials and Reagents

  1. Latex gloves
  2. 1.5 ml plastic tubes, sterile (Eppendorf, catalog number: 0030123328 )
  3. 1.0 ml pipette tips, sterile (Thermo Fisher Scientific, Thermo Scientific, catalog number: H-111-R100NS-Q )
  4. 0.2 ml pipette tips, sterile (Quality Scientific Plastics, Thermo Fisher Scientific, Thermo Scientific, catalog number: TTW110RS-Q )
  5. 10 ml Serological pipettes, sterile (As One, catalog number: 2-5237-04 )
  6. 50 ml Serological pipettes, sterile (As One, catalog number: 2-5237-06 )
  7. Bottle top 0.2-µm filter units, sterile (Thermo Fisher Scientific, Thermo Scientific, catalog number: 566-0020 )
  8. Combitips advanced 50 ml, sterile (Eppendorf, catalog number: 0030089480 )
  9. Petri dishes, sterile (Kord-Valmark, catalog number: 2901 )
  10. Quadrant Petri dishes, sterile (Kord-Valmark, catalog number: 2913 )
  11. Square Petri dishes with grids, sterile (Simport, catalog number: D210-16 )
  12. Wild-type C. elegans strain N2 (available at Caenorhabditis Genetics Center [CGC], https://cbs.umn.edu/cgc/home)
  13. E. coli OP50 (available at Caenorhabditis Genetics Center [CGC], https://cbs.umn.edu/cgc/home)
  14. 1-Nonanol (Sigma-Aldrich, catalog number: 131210-100ML )
  15. Ethanol (99.5%) (Wako Pure Chemical Industries, catalog number: 057-00451 )
  16. Chloroform (Nacalai Tesque, catalog number: 08401-65 )
  17. LB medium capsules (MP Biomedical, catalog number: 3002-021 )
  18. Sodium chloride (NaCl) (Nacalai Tesque, catalog number: 31320-05 )
  19. Bacto agar (BD, catalog number: 214010 )
  20. Bacto peptone (BD, catalog number: 211677 )
  21. Potassium dihydrogen phosphate (KH2PO4) (Nacalai Tesque, catalog number: 28721-55 )
  22. Di-potassium hydrogen phosphate (K2HPO4) (Nacalai Tesque, catalog number: 28726-05 )
  23. HEPES (Nacalai Tesque, catalog number: 17514-15 )
  24. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
  25. Calcium chloride dihydrate (CaCl2·2H2O) (Nacalai Tesque, catalog number: 06730-15 )
  26. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Nacalai Tesque, catalog number: 21003-75 )
  27. Potassium chloride (KCl) (Nacalai Tesque, catalog number: 28514-75 )
  28. D-sorbitol (Sigma-Aldrich, catalog number: S1876-1KG )
  29. Gelatin (Wako Pure Chemical Industries, catalog number: 073-06295 )
  30. Cholesterol (Wako Pure Chemical Industries, catalog number: 034-03002 )
  31. LB broth (see Recipe 1)
  32. NGM plates (see Recipe 2)
  33. 1.0 M potassium phosphate (pH 6.0) (see Recipe 3)
  34. 1.0 M HEPES-NaOH (pH 7.2) (see Recipe 4)
  35. 1.0 M CaCl2 (see Recipe 5)
  36. 1.0 M MgSO4 (see Recipe 6)
  37. Agar for resource localization assay plates (see Recipe 7)
  38. 2.0% Molten agar (see Recipe 8)
  39. 0.25% Aqueous gelatin solution (see Recipe 9)
  40. 5.0 mg/ml cholesterol (see Recipe 10)
  41. Chemotaxis assay plates (see Recipe 11)
  42. Doubly deionized water (ddH2O; see Recipe 12)

Equipment

  1. Safety goggles
  2. A laboratory coat
  3. Worm pick
  4. Dental burner (Phoenix-Dent, model: APT-3 )
  5. Bunsen burner (EISCO)
  6. Incubator (SANYO, model: MIR-553 )
  7. Heating magnetic stirrer (Thermo Fisher Scientific, Thermo Scientific, model: SP131324 )
  8. Magnetic stirrer bar
  9. 1.0 L beaker
  10. Pipet-Aid® XP (Drummond Scientific, model: Pipet-Aid® XP, catalog number: 4-000-101 )
  11. Multipette M4 (Eppendorf, catalog number: 4982000012 )
  12. P20 pipetman (Gilson, catalog number: F123600 )
  13. P100 pipetman (Gilson, catalog number: F123615 )
  14. P1000 pipetman (Gilson, catalog number: F123602 )
  15. Kimwipes S-200 (Nippon Paper Crecia, catalog number: 62011 )
  16. Osmometer (Gonotec, model: Osmomat 030-D )
  17. Autoclave (Tomy Digital Biology, model: SX-300 )
  18. Aspirator
  19. Stereomicroscope (Olympus, model: SZX16 )
  20. Water purification system (Merck, model: Elix® Essential 10 UV )
  21. Water purification system (Merck, model: Milli-Q® Synthesis A10® )

Procedure

Part I: Protocol for resource localization assay (the entire procedure is carried out on the bench)

  1. Preparation of synchronized worms
    1. Place 10 adult hermaphrodites on an OP50-seeded nematode growth medium (NGM) plate (see Recipe 2), 6 cm in diameter near the Bunsen burner flame, and incubate for 4 h at 20 °C.
    2. Remove adults from the NGM plates near the Bunsen burner flame, and incubate the plates for 4 days at 20 °C (Note 1).

  2. Preparation of resource localization assay plates
    1. Prepare agar with or without KCl (see Recipe 7).
    2. Pour agar without KCl, 13 ml each, into two diagonally located compartments in a quadrant dish.
    3. After solidifying, pour agar with KCl, 13 ml each, to each of two remaining compartments (Figures 1A and 1B) (Note 2).
    4. Leave the plate without lid at room temperature for 1 h to dry up the agar surface on the bench.
    5. Connect the compartments by placing a thin layer of 2.0% molten agar (see Recipe 8), using a P1000 pipetman with a blunted tip. Molten agar at 60 °C is poured along plastic separators from the center to the edge of a tilted plate (Figure 1C).
    6. Leave the plate at room temperature for 5 min before use to solidify the molten agar.


      Figure 1. Preparation of resource localization assay plates. A. A quadrant Petri dish poured with agar. KCl (+) indicates agar containing KCl. B. Side-view of the plate. Note that the agar surface is higher than the top of the plastic separator. C. Pouring of 2.0% molten agar on top of the plastic separator from the center to the edge along the separator.

  3. Resource localization assay
    1. Collect worms from an NGM culture plate, 6 cm in diameter, to a 1.5 ml tube by washing them off with 1.5 ml of 0.25% aqueous gelatin solution (see Recipe 9). After worms have settled at the bottom of the tube (< 1.0 min), remove most of the gelatin solution.
    2. Wash worms three times with 1.0 ml of 0.25% gelatin solution in the same way as above.
    3. Resuspend worms at the concentration of 50-100 worms in ~20 µl of gelatin solution.
    4. Place ~20 µl worm suspension at the center of a quadrant plate, using a P100 Pipetman with a blunted tip.
    5. Remove as much gelatin solution as possible with a Kimwipes wick.
    6. Put the lid on the plate, and incubate the plate for 30 min at room temperature.
    7. Place ~100 µl of chloroform on the lid, and then cover the chloroform with the agar plate to sacrifice worms (Video 1).

      Video 1. How to stop worm chemotaxis. At the end of chemotaxis assay, ~100 µl of chloroform was spread on the lid, and then the agar plate was placed upside down on the lid. The same procedure was used for the termination of chemotaxis in Part II.

    8. Count worms in each of the quadrants to calculate a performance index (PI) using the following equation (worms lying against the side of the plate and on the plastic separators are not counted):
      PI = [(# of worms in KCl compartments) - (# of worms in reference compartments)]/(# of total worms)

Part II: Protocol for chemotaxis assay using a square agar plate


  1. Preparation of synchronized worms as described in Part I.

  2. Chemotaxis assay on a square plate
    1. Draw lines on a square assay plate (see Recipe 11), 10 x 10 cm, as shown in Figure 2A.
    2. Prepare worm suspension, which contains ~50 worms per 10 µl, as described above in resource localization assay.
    3. Place ~10 µl each of the worm suspension at two places as shown in Figure 2A, using a P100 Pipetman with a blunted tip.
    4. Remove as much gelatin solution as possible with a Kimwipes wick.
    5. Wait for ~30 sec until worms start moving.
    6. Spot 3 µl each of 1.0% 1-nonanol diluted with ethanol at two places marked with ‘X’ as shown in Figure 2A, and immediately cover it with a lid.
    7. Incubate the plate for 10 min at room temperature, and then sacrifice worms with chloroform vapor as described above in resource localization assay.
    8. Count the number of worms in two areas with and without 1-nonanol to calculate a chemotaxis index (CI) using the following equation (worms lying against the side of the plate and on the lines are not counted):
      CI = [(# of worms in odor section) - (# of worms in reference)]/[(# of worms in odor section) + (# of worms in reference)]


      Figure 2. Chemotaxis away from 1-nonanol on a square agar plate. A. A square chemotaxis assay plate marked with red circles showing places where ~10 µl each of worm suspension was spotted, and with ‘X’ indicating positions where 1-nonanol was spotted. B. A typical example of chemotaxis assay after sacrificing worms with chloroform vapor. Note that most of worms (white dots) were repelled from 1-nonanol odor, and mainly located in the reference area on the assay plate.

Data analysis

Data analysis should be performed statistically [e.g., using Microsoft® Excel 2011 for Macintosh® with the add-in software Statcel3 (OMS Publ., Saitama, Japan)]. All data should be checked for normality of distribution and homogeneity of variance statistically (e.g., using χ2 goodness of fit test), and should also be evaluated statistically [e.g., using Student’s t-test for comparisons between pairs of groups, or one-way analysis of variance (ANOVA) for multiple comparisons between groups]. If ANOVA results are significant (P < 0.05), the Tukey-Kramer post hoc test or equivalent is used. Results are reported as mean ± the standard error of the mean. The sensitivity of wild-type worms to various concentrations of KCl and 1-nonanol is examined by resource localization assay and chemotaxis assay using square plates, respectively, and is shown in Figure 3.


Figure 3. Sensitivity of wild-type C. elegans to various concentrations of KCl and 1-nonanol. A. Performance index (PI) values of wild-type worms, which were measured on resource localization assay plates containing agar with KCl (5-200 mM) or without KCl (n = 6-9 assays). B. Chemotaxis index (CI) values of wild-type worms away from 1-nonanol variously diluted with ethanol, which were assayed using square agar plates (n = 9-15 assays). Asterisks indicate statistically significant (*P < 0.05, **P < 0.01) differences determined by one-way ANOVA, followed by the Tukey-Kramer test for further pair-wise comparisons of all data points. These data are modified from Nishijima and Maruyama (2017). 

Notes

  1. It is easier to remove adult worms from plates by using an aspirator equipped with a 0.2 ml pipette tip than using a worm picker.
  2. It is convenient to fill quadrant compartments with agar by using a repeating pipette such as Multipette M4. In case that poured agar fused with that in an adjacent compartment, discard the plate since chemicals diffuse out to its adjacent agar.
  3. 13 ml of agar is required for filling each of four compartments of a quadrant plate, 10 cm in diameter. In this recipe, we describe how to make 500 ml of agar containing 100 mM KCl. Sorbitol was added to reference agar without KCl to give the same osmolality to 100 mM KCl. The osmolality of solutions was measured using an osmometer.

Recipes

  1. LB broth
    1. Dissolve 25 capsules of LB medium (see Materials and Reagents) in 1.0 L ddH2O and autoclave at 121 °C for 20 min
    2. Store at room temperature
  2. NGM plates
    1. Dissolve 3 g NaCl, 20 g agar and 2.5 g Bacto peptone in 972 ml doubly deionized water (ddH2O) and autoclave at 121 °C for 20 min
    2. Let the medium cool to ~60 °C on a heating magnetic stirrer
    3. Under sterile condition, add 25 ml 1.0 M potassium phosphate (pH 6.0), 1.0 ml 1.0 M CaCl2, 1.0 ml 1.0 M MgSO4 and 1.0 ml 5.0 mg/ml cholesterol in ethanol
    4. Pour 8 ml of the NGM agar into a Petri dish, 6 cm in diameter, and leave the plate overnight at room temperature
    5. Prepare an overnight culture of OP50 with LB broth (see Recipe 1), and keep the culture on ice until use
    6. Spread ~0.1 ml of OP50 culture on the NGM plate under sterile condition, and incubate overnight at room temperature
  3. 1.0 M potassium phosphate (pH 6.0)
    1. Dissolve 35.6 g K2HPO4 and 108.3 g KH2PO4 in ddH2O, and adjust the volume to 1.0 L
    2. After autoclaving at 121 °C for 20 min, store the solution at room temperature
  4. 1.0 M HEPES-NaOH (pH 7.2)
    1. Immediately before use, prepare 1.0 N NaOH by dissolving 12 g NaOH pellets in 300 ml ddH2O
    2. Dissolve 238.3 g HEPES in ddH2O, and adjust to pH 7.2 with 251 ml 1.0 N NaOH
    3. Adjust the volume to 1.0 L by adding ddH2O, followed by filtration with a 0.22-µm filter
    4. Store the solution at room temperature
  5. 1.0 M CaCl2
    1. Dissolve 147 g CaCl2·2H2O in ddH2O, and adjust the volume to 1.0 L
    2. After autoclaving at 121 °C for 20 min, store at room temperature
  6. 1.0 M MgSO4
    1. Dissolve 246.4 g MgSO4·7H2O in ddH2O, and adjust the volume to 1.0 L
    2. After autoclaving at 121 °C for 20 min, store at room temperature
  7. Agar for resource localization assay plates (Note 3)
    1. Prepare agar medium containing 100 mM KCl by dissolving 3.7 g KCl and 10 g agar in 494 ml ddH2O by autoclaving at 121 °C for 20 min
    2. Prepare agar medium without KCl by dissolving 92 ml 1.0 M sorbitol and 10 g agar in 402 ml ddH2O by autoclaving at 121 °C for 20 min
    3. Let the agar media cool to ~60 °C on a heating magnetic stirrer
    4. Add 5 ml 1.0 M HEPES-NaOH (pH 7.2), 0.5 ml 1.0 M CaCl2 and 0.5 ml 1.0 M MgSO4 while heating
  8. 2.0% Molten agar
    1. Dissolve 2 g agar in 100 ml ddH2O by autoclaving
    2. Let the solution cool to ~60 °C on a heating magnetic stirrer
  9. 0.25% Aqueous gelatin solution
    1. Dissolve 2.5 g gelatin in 1.0 L ddH2O
    2. After autoclaving at 121 °C for 20 min, store at room temperature
  10. 5.0 mg/ml cholesterol
    1. Dissolve 250 mg cholesterol in 50 ml of ethanol
    2. Store at room temperature
  11. Chemotaxis assay plates
    1. Dissolve 15 g agar in 993 ml ddH2O by autoclaving
    2. Let the solution cool to ~60 °C on a heating magnetic stirrer
    3. Add 5.0 ml 1.0 M potassium phosphate (pH 6.0), 1.0 ml 1.0 M CaCl2, and 1.0 ml 1.0 M MgSO4
    4. Pour 14 ml of the solution into a square Petri dish, 10 x 10 cm, and leave the dish on bench overnight
  12. Doubly deionized water
    First, treat tap water with a Millipore Elix 10 UV water purification system, and then with a Millipore Milli-Q Synthesis A10 water purification system

Acknowledgments

A brief version of this protocol was described in Nishijima and Maruyama (2017). This work was partly supported by a JSPS grant (16K07007 to T.M.) and funding to the Information Processing Biology Unit from the Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan. The authors hereby declare no conflict of interest or competing interest.

References

  1. Amano, H. and Maruyama, I. N. (2011). Aversive olfactory learning and associative long-term memory in Caenorhabditis elegans. Learn Mem 18(10): 654-665.
  2. Bargmann, C.I., Hartwieg, E. and Horvitz, H.R. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74(3): 515-527.
  3. Bargmann, C. I. and Horvitz, H. R. (1991). Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans. Neuron 7(5): 729-742.
  4. Dusenbery, D. B. (1974). Analysis of chemotaxis in the nematode Caenorhabditis elegans by countercurrent separation. J Exp Zool 188(1): 41-47.
  5. Jansen, G., Weinkove, D. and Plasterk, R. H. A. (2002). The G-protein gamma subunit gpc-1 of the nematode C.elegans is involved in taste adaptation. EMBO J 21(5): 986-994.
  6. Murayama, T. and Maruyama, I. N. (2013). Decision making in C. elegans chemotaxis to alkaline pH: Competition between two sensory neurons, ASEL and ASH. Commun Integr Biol 6(6): e26633.
  7. Nishijima, S. and Maruyama, I. N. (2017). Appetitive Olfactory Learning and Long-Term Associative Memory in Caenorhabditis elegans. Front Behav Neurosci 11: 80.
  8. Ortiz, C. O., Faumont, S., Takayama, J., Ahmed, H. K., Goldsmith, A. D., Pocock, R., McCormick, K. E., Kunimoto, H., Iino, Y., Lockery, S. and Hobert, O. (2009). Lateralized gustatory behavior of C. elegans is controlled by specific receptor-type guanylyl cyclases. Curr Biol 19(12): 996-1004.
  9. Sassa, T., Murayama, T. and Maruyama, I. N. (2013). Strongly alkaline pH avoidance mediated by ASH sensory neurons in C. elegans. Neurosci Lett 555: 248-252.
  10. Troemel, E. R., Kimmel, B. E. and Bargmann, C. I. (1997). Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans. Cell 91(2): 161-169.
  11. Ward, S. (1973). Chemotaxis by the nematode Caenorhabditis elegans: Identification of attractants and analysis of response by use of mutants. Proc Natl Acad Sci USA 70(3): 817-821.
  12. Wicks, S. R., de Vries, C. J., van Luenen, H. G. A. M. and Plasterk, R. H. A. (2000). CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans. Dev Biol 221(2): 295-307.

简介

线虫<秀秀隐杆线虫广泛用于从简单的化学感应到联想学习和记忆的行为研究。 这些研究对于确定吸引和厌恶化学物质的最佳浓度至关重要。 线虫可以感觉到。 在这里我们描述了一种资源定位测定法,其中将感兴趣的化学化合物置于象限板的两个隔室中以确定化学品在行为研究中的最佳浓度。 使用该测定,我们确定了水溶性引诱剂KCl的最佳浓度,作为研究关联学习和记忆的无条件刺激。 在这个协议中,我们还描述了一个趋化性测定,使用一个方形琼脂平板上点着一种厌恶的嗅觉信号1-壬醇作为条件刺激。

【背景】线虫线虫广泛用作动物行为研究的模式生物。 ℃。秀丽隐杆线虫感知主要由最大的化学感受器官(Ward,1973; Dusenbery,1974; Bargmann和Horvitz,1991; Bargmann等人)等介导的各种水溶性和挥发性化学物质。 1993)。行为研究必须确定可以被感知的化学物质的精确浓度。线虫。为了确定用于C的水溶性引诱剂的最佳浓度。线虫,Wicks等人(2000)使用象限琼脂平板进行行为测定,其中感兴趣的化学物质与琼脂在两个隔室中混合,并且该测定已广泛用于许多化学品(例如Jansen等人,2002; Ortiz等人,2009; Murayama和Maruyama,2013; Sassa&lt; em&gt; et al。,2013)。趋化性分析也被用于测量C的敏感性。 (Bargmann等人,1993; Troemel等人,1997)上的挥发性化合物。 ℃。线虫也是研究关联学习和记忆的优秀模式生物,其中水溶性化学物质和挥发性化学物质被用作无条件刺激(US)和条件刺激(CS)(Amano和Maruyama, 2011年;西岛和丸山,2017年)。为了有效地调节蠕虫,CS和US的浓度是关键参数。使用象限琼脂平板的资源定位测定和方形琼脂平板上的趋化性测定成功用于确定用于学习和记忆研究的US和CS的最佳浓度。因此,这些测定法可以用于许多其他有吸引力和排斥性的化学物质。线虫行为分析。

关键字:引诱, 厌恶刺激, 化学感受行为, 学习和记忆, 平板分析法

材料和试剂

  1. 乳胶手套
  2. 1.5毫升塑料管,无菌(Eppendorf,目录号:0030123328)
  3. 1.0 ml移液枪头,无菌(Thermo Fisher Scientific,Thermo Scientific,目录号:H-111-R100NS-Q)
  4. 0.2 ml移液枪头,无菌(Quality Scientific Plastics,Thermo Fisher Scientific,Thermo Scientific,目录号:TTW110RS-Q)
  5. 10毫升血清移液管,无菌(As One,产品目录号:2-5237-04)
  6. 50毫升血清移液器,无菌(As One,目录号:2-5237-06)
  7. 无菌(Thermo Fisher Scientific,Thermo Scientific,产品目录号:566-0020)的瓶顶部0.2-μm过滤器单元
  8. Combitips先进的50毫升,无菌(Eppendorf,目录号:0030089480)
  9. 培养皿,无菌(Kord-Valmark,目录号:2901)
  10. 象限培养皿,无菌(Kord-Valmark,目录号:2913)
  11. 带网格的正方形培养皿,无菌(Simport,目录号:D210-16)
  12. 野生型 C。 eleg ans菌株N2(可在Caenorhabditis遗传中心[CGC]处获得, https://cbs.umn .edu / cgc / home
  13. 电子。大肠杆菌OP50(可在Caenorhabditis Genetics Center [CGC]处获得, https://cbs.umn.edu / cgc / home
  14. 1-壬醇(Sigma-Aldrich,目录号:131210-100ML)
  15. 乙醇(99.5%)(Wako Pure Chemical Industries,目录号:057-00451)
  16. 氯仿(Nacalai Tesque,目录号:08401-65)
  17. LB培养基胶囊(MP Biomedical,目录号:3002-021)
  18. 氯化钠(NaCl)(Nacalai Tesque,目录号:31320-05)
  19. 细菌琼脂(BD,目录号:214010)
  20. 细菌蛋白胨(BD,目录号:211677)
  21. 磷酸二氢钾(KH 2 PO 4)(Nacalai Tesque,目录号:28721-55)
  22. 磷酸二氢钾(KH 2 HPO 4)(Nacalai Tesque,目录号:28726-05)
  23. HEPES(Nacalai Tesque,目录号:17514-15)
  24. 氢氧化钠(NaOH)(Wako Pure Chemical Industries,目录号:198-13765)
  25. 氯化钙二水合物(CaCl 2•2H 2 O)(Nacalai Tesque,目录号:06730-15)
  26. 硫酸镁七水合物(MgSO 4•7H 2 O)(Nacalai Tesque,目录号:21003-75)
  27. 氯化钾(KCl)(Nacalai Tesque,目录号:28514-75)
  28. D-山梨醇(Sigma-Aldrich,目录号:S1876-1KG)
  29. 明胶(Wako Pure Chemical Industries,目录号:073-06295)
  30. 胆固醇(Wako Pure Chemical Industries,目录号:034-03002)
  31. LB肉汤(参见配方1)
  32. NGM板(见配方2)
  33. 1.0M磷酸钾(pH 6.0)(见配方3)
  34. 1.0 M HEPES-NaOH(pH 7.2)(见配方4)
  35. 1.0 M CaCl 2(见方案5)
  36. 1.0M MgSO 4(参见配方6)
  37. 用于资源定位测定板的琼脂(见第7部分)
  38. 2.0%熔琼脂(见方法8)
  39. 0.25%明胶水溶液(见配方9)
  40. 5.0毫克/毫升胆固醇(见方案10)
  41. 趋化性试验平板(见11)
  42. 双去离子水(ddH2O:参见12)

设备

  1. 安全护目镜
  2. 实验室外套
  3. 蠕虫捡
  4. 牙科燃烧器(Phoenix-Dent,型号:APT-3)
  5. 本生灯(EISCO)
  6. 孵化器(SANYO,型号:MIR-553)
  7. 加热磁力搅拌器(Thermo Fisher Scientific,Thermo Scientific,型号:SP131324)
  8. 磁力搅拌棒
  9. 1.0 L烧杯
  10. Pipet-Aid XP(Drummond Scientific,型号:Pipet-Aid XP,目录号:4-000-101)
  11. Multipette M4(Eppendorf,目录号:4982000012)
  12. P20 pipetman(Gilson,目录号:F123600)
  13. P100移液枪(Gilson,目录号:F123615)
  14. P1000移液器(Gilson,目录号:F123602)
  15. Kimwipes S-200(Nippon Paper Crecia,目录号:62011)
  16. 渗压计(Gonotec,型号:Osmomat 030-D)
  17. 高压灭菌器(Tomy Digital Biology,型号:SX-300)
  18. 吸气器
  19. 立体显微镜(奥林巴斯,型号:SZX16)
  20. 水净化系统(Merck,型号:Elix Essential 10 UV)
  21. 水净化系统(Merck,型号:Milli-Q合成A10))

程序

第一部分:资源定位分析方案<整个程序是在实验台上进行的)

  1. 准备同步蠕虫
    1. 将10个成年雌雄同体放在OP50接种的线虫生长培养基(NGM)平板上(见方案2),直径6厘米,靠近本生灯火焰,并在20℃孵育4小时。
    2. 从本生灯火焰附近的NGM板上移除成虫,在20°C孵育板4天(注1)。

  2. 制备资源定位测定板
    1. 准备有或没有氯化钾的琼脂(见第7部分)。

    2. 将没有氯化钾的琼脂倒入每个13毫升的琼脂培养皿中,放入象限培养皿中对角放置的两个隔间。
    3. 固化后,将琼脂用每个13 ml的KCl倒入剩余的两个隔室(图1A和1B)(注2)。

    4. 在室温下将盖板放在无盖的位置1小时,以便将工作台上的琼脂表面干燥。
    5. 通过放置一层薄薄的2.0%熔融琼脂(参见配方8),使用带钝端的P1000移液管连接隔室。将60°C的熔琼脂沿着塑料分离器从中心倾斜到倾斜板的边缘(图1C)。

    6. 在使用前将板放置在室温下5分钟以固化熔融琼脂。


      图1.制备资源定位测定板。 :一种。一个象限培养皿倒入琼脂。 KCl(+)表示含有KCl的琼脂。 B.板的侧视图。请注意,琼脂表面高于塑料分离器的顶部。 C.将塑料分离器顶部的2.0%熔融琼脂从中心注入沿分离器的边缘。

  3. 资源定位测定
    1. 通过用1.5ml 0.25%明胶水溶液(参见配方9)将它们从直径6cm的NGM培养板收集到1.5ml管中。蠕虫在管的底部沉降(<1.0分钟)后,除去大部分明胶溶液。
    2. 以上述相同的方式用1.0ml的0.25%明胶溶液清洗蠕虫三次。

    3. 在〜20μl明胶溶液中以50-100蠕虫浓度重新悬浮蠕虫。

    4. 使用P100移液器和钝头,在一个象限板的中心放置〜20μl蠕虫悬浮液。

    5. 用Kimwipes灯芯尽可能多地去除明胶溶液。
    6. 将盖子放在平板上,在室温下孵育30分钟。
    7. 将约100μl氯仿置于盖上,然后用琼脂板覆盖氯仿以牺牲蠕虫(视频1)。

      视频1

    8. 计算每个象限中的蠕虫以使用以下公式计算性能指数(PI)(蠕虫位于平板侧面和塑料分离器上不计算在内):
      PI = [(KCl隔间中的蠕虫数量) - (参考隔间中的蠕虫数量)] /(蠕虫总数)

第二部分:使用方形琼脂平板进行趋化性分析的方案


  1. 准备第一部分描述的同步蠕虫。

  2. 在方形板上进行趋化性测定
    1. 如图2A所示,在方形测定板上画线(见11),10 x 10 cm。
    2. 如上文资源定位测定中所述,准备蠕虫悬浮液,每10μl含有约50个蠕虫。
    3. 如图2A所示,将两个蠕虫悬浮液放置在两个位置,使用P100移液器和钝头。

    4. 用Kimwipes灯芯尽可能多地去除明胶溶液。
    5. 等待约30秒,直到蠕虫开始移动。
    6. 如图2A所示,将3%的1.0%1-壬醇用乙醇稀释至两个标有'X'的地方,并立即盖上盖子。
    7. 在室温下将平板温育10分钟,然后如上所述在资源定位测定中用氯仿蒸气处理蠕虫。
    8. 计算两个区域中含有或不含1-壬醇的蠕虫数量,使用以下公式计算趋化性指数(CI)(蠕虫位于平板侧面并且不在线上):
      CI = [(臭气部分中的蠕虫数量) - (参考中的蠕虫数量)] / [(臭气部分中的蠕虫数量)+(参考中的蠕虫数量)]


      图2.在方形琼脂平板上远离1-壬醇的趋化性A.用红色圆圈标记的方形趋化性测定板显示其中发现约10μl各种蠕虫悬浮液的位置,并且用'X '表示1-壬醇被点出的位置。 B.用氯仿蒸气杀死蠕虫后的趋化性分析的典型例子。请注意,大部分蠕虫(白色圆点)都被1-壬醇气味排斥,主要位于检测板上的参考区域。

数据分析

数据分析应该使用附带软件Statcel3(OMS Publ。,Inc。)使用Microsoft > Excel 2011 for Macintosh ®进行统计学分析[ eg 埼玉,日本)]。所有数据均应检查分布的正态性和统计方差的均匀性(例如,使用 2 适合性测试),并且还应该进行统计学评估[例如,使用Student's t -test进行组间比较,或单因素方差分析(ANOVA)多组比较]。如果方差分析结果显着(<0.05),则使用Tukey-Kramer事后检验或等效方法。结果报告为平均值±平均值的标准误差。野生型蠕虫对不同浓度的KCl和1-壬醇的敏感性分别通过资源定位测定法和使用正方形平板的趋化性测定法进行检查,结果如图3所示。


图3.野生型 C的敏感性。线虫到各种浓度的KCl和1-壬醇。 :一种。野生型蠕虫的性能指数(PI)值是在含有KCl(5-200 mM)或不含KCl的琼脂的资源定位测定板上测量的( n = em = 6-9分析)。 B.用乙醇不同稀释的野生型蠕虫远离1-壬醇的趋化性指数(CI)值,其使用方形琼脂平板(n = 9-15分析)测定。星号表示通过单因素ANOVA确定的统计学显着差异(* P <0.05,** P <0.01)差异,然后通过Tukey-Kramer测试进一步确定对所有数据点的比较。这些数据由西岛和丸山(2017)修改。&nbsp;

笔记

  1. 使用装有0.2毫升枪头的吸气器比使用蠕虫捡拾器更容易从盘子上除去成虫。
  2. 通过使用Multipette M4等重复移液器,用琼脂填充象限室很方便。如果倒入的琼脂与相邻隔室中的琼脂融合,则丢弃该板,因为化学品会扩散到其相邻的琼脂上。
  3. 需要13ml琼脂来填充直径10cm的象限板的四个隔室中的每一个。在这个配方中,我们描述了如何制作500毫升含100毫米氯化钾的琼脂。将山梨醇添加至不含KCl的参照琼脂以赋予与100mM KCl相同的渗透压。
    使用渗压计测量溶液的重量克分子渗透浓度

食谱

  1. LB肉汤
    1. 将1.0L ddH 2 O中的25个LB培养基(参见材料和试剂)溶解并在121℃高压灭菌20分钟。
    2. 在室温下储存
  2. NGM板
    1. 在972ml双去离子水(ddH2O)中溶解3g NaCl,20g琼脂和2.5g细菌蛋白胨,并在121℃高压灭菌20分钟。
    2. 使用加热磁力搅拌器将介质冷却至〜60°C
    3. 在无菌条件下,加入25ml 1.0M磷酸钾(pH 6.0),1.0ml 1.0M CaCl 2,1.0ml 1.0M MgSO 4和1.0ml 5.0mg / ml在乙醇中的胆固醇
    4. 将8毫升NGM琼脂倒入直径6厘米的陪替氏培养皿中,并在室温下过夜。
    5. 用LB肉汤准备过夜培养的OP50培养物(见方法1),并将培养物保存在冰上直至使用

    6. 在无菌条件下将约0.1ml的OP50培养物铺展在NGM板上,并在室温下孵育过夜
  3. 1.0M磷酸钾(pH 6.0)
    1. 在ddH 2溶液中溶解35.6g K 2 HPO 4 4和108.3g KH 2 PO 4 4, O,并将音量调整到1.0 L
    2. 在121°C高压灭菌20分钟后,将溶液置于室温下
  4. 1.0 M HEPES-NaOH(pH 7.2)
    1. 在即将使用之前,通过将12g NaOH丸粒溶解在300ml ddH 2 O中制备1.0N NaOH
    2. 在ddH 2 O中溶解238.3g HEPES,并用251ml 1.0N NaOH调节至pH 7.2。
    3. 加入ddH <2> O,将体积调节至1.0 L,然后用0.22μm过滤器过滤。

    4. 在室温下储存溶液
  5. 1.0 M CaCl 2 2
    1. 在ddH 2 O中溶解147g CaCl 2•2H 2 O,并将体积调节至1.0L
    2. 在121°C高压灭菌20分钟后,在室温下保存
  6. 1.0M MgSO 4
    1. 在ddH 2 O中溶解246.4g MgSO 4•7H 2 O,并将体积调节至1.0L。
    2. 在121°C高压灭菌20分钟后,在室温下保存
  7. 琼脂用于资源定位测定板(注3)
    1. 通过在121℃下高压灭菌20分钟,将3.7g KCl和10g琼脂溶解于494ml ddH 2 O中,制备含有100mM KCl的琼脂培养基。
    2. 通过在121℃下高压灭菌20分钟,将92ml 1.0M山梨糖醇和10g琼脂溶解在402ml ddH 2 O中,制备不含KCl的琼脂培养基。
    3. 让琼脂培养基在加热磁力搅拌器上冷却至〜60°C
    4. 在加热的同时加入5ml 1.0M HEPES-NaOH(pH7.2),0.5ml 1.0M CaCl 2和0.5ml 1.0M MgSO 4。
  8. 2.0%熔琼脂
    1. 通过高压灭菌法将2g琼脂溶解在100ml ddH 2 O中
    2. 用加热磁力搅拌器将溶液冷却至〜60°C
  9. 0.25%明胶水溶液
    1. 在1.0L ddH 2 O中溶解2.5g明胶
    2. 在121°C高压灭菌20分钟后,在室温下保存
  10. 5.0毫克/毫升胆固醇
    1. 将250毫克胆固醇溶解在50毫升乙醇中
    2. 在室温下储存
  11. 趋化性测定板
    1. 通过高压灭菌将15g琼脂溶解在993ml ddH 2 O中
    2. 用加热磁力搅拌器将溶液冷却至〜60°C
    3. 加入5.0ml 1.0M磷酸钾(pH 6.0),1.0ml 1.0M CaCl 2和1.0ml 1.0M MgSO 4。
    4. 将14毫升溶液倒入10×10厘米的方形培养皿中,并将培养皿放在长凳上过夜。
  12. 双去离子水
    首先,用Millipore Elix 10紫外线水净化系统处理自来水,然后用Millipore Milli-Q Synthesis A10水净化系统处理自来水。

致谢

在西岛和丸山(2017)中描述了该协议的简要版本。这项工作部分得到了日本冲绳县科学技术研究生院冲绳研究所信息处理生物部资助的JSPS资助(16K07007至T.M.)和资助。作者在此声明不存在利益冲突或利益冲突。

参考

  1. Amano,H。和Maruyama,I.N。(2011)。 在线虫线虫中厌恶嗅觉学习和联想长期记忆 学习Mem 18(10):654-665。
  2. Bargmann,C.I.,Hartwieg,E。和Horvitz,H.R.(1993)。 恶臭选择性基因和神经元介导线虫中的嗅觉。< / a> Cell 74(3):515-527。
  3. Bargmann,C.I。和Horvitz,H.R。(1991)。 具有重叠功能的化学感受神经元可直接对多种化学物质产生趋化性。 elegans 。 Neuron 7(5):729-742。
  4. Dusenbery,D.B。(1974)。 通过逆流分离分析线虫中的趋化性 a> J Exp Zool 188(1):41-47。
  5. Jansen,G.,Weinkove,D。和Plasterk,R.H.A。(2002)。 线虫线虫的G蛋白γ亚基gpc-1 >参与品味适应。 EMBO J 21(5):986-994。
  6. Murayama,T。和Maruyama,I.N。(2013)。 决策制定在 C。线虫趋化性至碱性pH:两种感觉神经元之间的竞争,ASEL和ASH。 Commun Integr Biol 6(6):e26633。
  7. Nishijima,S。和Maruyama,I.N.(2017)。 线虫中的嗜食性嗅觉学习和长期联想记忆 >。
    。 Frontfev Neurosci 11:80。
  8. Ortiz,CO,Faumont,S.,Takayama,J.,Ahmed,HK,Goldsmith,AD,Pocock,R.,McCormick,KE,Kunimoto,H.,Iino,Y.,Lockery,S.和Hobert,O. (2009年)。 C的横向味觉行为。 elegans 由特定的受体型鸟苷酸环化酶控制。 19(12):996-1004。
  9. Sassa,T.,Murayama,T。和Maruyama,I.N。(2013)。 由ASH感觉神经元介导的强碱性pH避免。 elegans 。 Neurosci Lett 555:248-252。
  10. Troemel,E.R.,Kimmel,B.E。和Bargmann,C.I。(1997)。 重编程趋化反应:感觉神经元定义嗅觉中的嗅觉偏好。 elegans 。 Cell 91(2):161-169。
  11. Ward,S。(1973)。 线虫趋化秀丽隐杆线虫:诱剂的鉴定和反应分析(Proc Natl Acad Sci USA)70(3):817-821。
  12. Wicks,S.R.,de Vries,C. J.,van Luenen,H. G. A. M.和Plasterk,R. H. A.(2000)。 CHE-3是一种胞质动力蛋白重链,对于感官纤毛结构和功能是必需的 Caenorhabditis elegans 。 Dev Biol 221(2):295-307。
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引用:Murayama, T. and Maruyama, I. N. (2018). Plate Assay to Determine Caenorhabditis elegans Response to Water Soluble and Volatile Chemicals. Bio-protocol 8(4): e2740. DOI: 10.21769/BioProtoc.2740.
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