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Imaging the Pharynx to Measure the Uptake of Doxorubicin in Caenorhabditis elegans
咽部成像测定秀丽隐杆线虫中多柔比星的摄取   

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Abstract

Caenorhabditis elegans offers an array of advantages to investigate the roles of uptake transporters. Herein, an epifluorescent microscopy approach was developed to monitor the uptake of the autofluorescent anticancer drug, doxorubicin, into the pharynx of C. elegans by organic cation transporters.

Keywords: C. elegans(秀丽隐杆线虫), Organic cation uptake transporters(有机阳离子吸收转运蛋白), Drug uptake(药物摄取), Doxorubicin(多柔比星), Autofluorescent drug(自发荧光药物), Epifluorescent microscopy(落射荧光显微镜检查), RNAi feeding bacteria(RNAi喂养细菌)

Background

Human cells have over 450 solute carrier transporters that are believed to facilitate the uptake of several ions, nutrients, as well as both therapeutic and anticancer drugs (Cesar-Razquin et al., 2015). However, the roles and substrates of a large number of these uptake transporters are not known. C. elegans is an inexpensive model organism that offers a multitude of advantages over mammalian cells to rapidly study many biological processes that are highly conserved in nature. During the last decade, this organism has been instrumental in several drug discovery programs to identify novel small molecules, e.g., those that act as antimicrobials and inhibit oxidative stress, although the yield of bioactive compounds has been less striking (Burns et al., 2010; O’Reilly et al., 2014). We reason that the recovery rate could be higher if there is greater and selective influx of the molecules by uptake transporters into the animal cells. To date, only three studies have been performed to understand the roles of uptake transporters in C. elegans (Wu et al., 1999; Cheah et al., 2013; Papaluca and Ramotar, 2016). Thus, characterization of the function and substrate specificities of uptake transporters in this organism will be advantageous towards improving the strategies employed to identify novel bioactive molecules. Herein, we outline a method to monitor uptake of the anticancer drug doxorubicin into the pharynx of C. elegans (Papaluca and Ramotar, 2016). Doxorubicin autofluorescence can be readily monitored by several widely available detection systems such as the epifluorescent microscope. We note that several benefits can be derived from this approach including a hunt for novel therapeutic substrates of the transporter by competing for doxorubicin uptake.

Materials and Reagents

  1. Petri dish 60 x 15 mm (SARSTEDT, catalog number: 82.1194.500 )
  2. 15 ml conical tube
  3. Frosted microscope slides (size: 1 x 3” ; thickness: 1-2 mm) (UltiDent Scientific, catalog number: 170-7107A )
  4. Microscope cover glass (size: 22 x 22 #1.5) (Fisher Scientific, catalog number: 12-541B )
  5. Platinum wire (Thomas Scientific, catalog number: 1233S71 )
  6. Pasteur pipet (Fisher Scientific, catalog number: 13-678-20C )
  7. E. coli bacteria HT115DE3 with the plasmid pL4440-empty vector
  8. E. coli bacteria HT115DE3 with the plasmid pL4440-oct-1 (Ahringer’s collection). Sequence verified
  9. E. coli bacteria HT115DE3 with the plasmid pL4440-oct-2 (Ahringer’s collection). Sequence verified
  10. Bristol N2 (wild type) and RB1084 [oct-1(ok1051) I] from Caenorhabditis Genetic Center
  11. Ampicillin (Sigma-Aldrich, catalog number: A9518 )
  12. Potassium phosphate monobasic (KH2PO4) (Bio Basic, catalog number: PB0445 )
  13. Magnesium sulfate (MgSO4) (Bio Basic, catalog number: MRB0329 )
  14. Calcium chloride dihydrate (CaCl2) (Fisher Scientific, catalog number: C79-500 )
  15. Cholesterol (Sigma-Aldrich, catalog number: C8503 )
  16. Ethanol 100% (works from any company)
  17. Doxorubicin (for Research from Hôpital Maisonneuve-Rosemont, Montreal, Canada). Stock concentration at 2 mg/ml
  18. IPTG (Bio Basic, catalog number: PRB0447 )
  19. Levamisol hydrochloride (MP Biomedical, catalog number: 155228 )
  20. Clear nail polish from Wild Shine from Dollarama
  21. Tryptone (Bio Basic, catalog number: TG217 (G211)) for Luria Broth (LB) media
  22. Yeast extract (Wisent Bioproducts, catalog number: 800-150-LG ) for LB media
  23. Sodium chloride (NaCl) (Wisent Bioproducts, catalog number: 600-082 )
  24. Bacteriological agar (Wisent Bioproducts, catalog number: 800-010-CG )
  25. Peptone (Wisent Bioproducts, catalog number: 800-157-LG ) for nematode grown media (NGM)
  26. Agarose (Wisent Bioproducts, catalog number: 800-015-CG )
  27. Sodium hydroxide (NaOH) (Bio Basic, catalog number: SB0617 )
  28. Bleach Lavo Pro6 (Lavo Inc, Montreal, Canada)
  29. Sodium phosphate dibasic (Na2HPO4) (Bio Basic, catalog number: S0404 )
  30. LB solution (see Recipes)
  31. Nematode growth media (NGM) (see Recipes)
  32. Agar pad (see Recipes)
  33. Alkaline Hypochlorite solution for bleaching the worms (see Recipes)
  34. M9 buffer (see Recipes)

Equipment

  1. Incubator at 20 °C, but with a range from 15 to 37 °C (SHEL LAB, model: 2020 )
  2. 37 °C incubator (Panasonic Healthcare, model: Mir-262 )
  3. 37 °C shaker (Inforst, model: Multitron Standard )
  4. 500 ml glass bottle (Wheaton graduated glass media bottles with lined caps)
  5. Microwave (inverter model, Panasonic Healthcare)
  6. 55 °C water bath (Precision Scientific, catalog number: 66800 )
  7. Metal spatula (VWR)
  8. Flame
  9. Neutrex culture tubes 16 x 15 mm
  10. Pyrex Erlenmeyer flask different sizes for bacteria culturing
  11. Autoclave
  12. Stereomicroscope Leica MZ 8 (Leica 10445538 Plan Microscope Objective Lens 1.0x) (Leica, model: MZ 8 )
  13. DeltaVision Elite Restoration System (GE Healthcare, model: DeltaVision Elite High Resolution Microscope ) and the DeltaVision imaging system user’s manual
  14. Fisher Vortex Genie 2 (Fisher Scientific, catalog number: 12-812 )
  15. Eppendorf 5810 R centrifuge (Eppendorf, model: 5810 R )
  16. VWR rocking platform shakers basic (VWR)
  17. Ptc-100 Programmable Thermal Controller 96 Well (Bio-Rad Laboratories, model: Ptc-100® Programmable Thermal Controller )
  18. Centrifuge (Sigma Centrifuge, model: Sigma 1-14 )

Software

  1. ImageJ imaging software
  2. SoftWorx software

Procedure

  1. Preparation of bacteria and media for worm growth
    1. Bacteria preparation (C. elegans food)
      1. In 10 ml LB medium, add 10 µl ampicillin (stock 100 mg/ml).
      2. Inoculate a few colonies of E. coli HT115DE3 empty vector bacteria into the LB + ampicillin medium.
        Note: Alternative bacteria: E. coli OP50.
      3. Incubate for 6 h at 37 °C in an orbital shaker at 200 rpm.
    2. C. elegans growth media preparation
      1. Melt the solid NGM contained in a glass bottle (500 ml) using a microwave (power level 2 for 25 min).
      2. Keep the melted medium at 55 °C in a water bath for 15-20 min.
      3. Before usage, add:
        12.5 ml of 1 M KH2PO4 pH 6
        500 µl of 1 M MgSO4
        500 µl of 1 M CaCl2
        500 µl cholesterol (stock 5 mg/ml in 95% ethanol)
        500 µl of ampicillin (stock 100 mg/ml), if using knockdown bacteria strain HT115DE3 carrying the L4440 vector or the target gene.
      4. Gently invert the bottle a few times to mix all the ingredients and then pour 6 ml of the medium into each of 60 x 15 mm Petri dish.
      5. Let it dry at room temperature for around 1 h.
      6. Once dried, using a sterilized bacterial plating rod spread 70 µl of bacteria prepared in step A1 above to form a lawn. The optical density OD600 of the bacteria is approximately 1.0. Alternatively, the bacteria can be added as 3 drops distributed onto the plate.
      7. Incubate the plate overnight at 37 °C.
    3. Worm growth
      1. From a grown worm plate, cut 4 pieces of approximately 1 x 1 cm agar with a pointy spatula sterilized with 100% ethanol and flame.
      2. Transfer each agar piece by turning them over onto each of 4 plates prepared in step A2.
      3. Incubate these worm plates for 2 days at 20 °C.

  2. Worm synchronisation
    1. The 4 plates are expected to be filled with adult worms, visualized through a stereomicroscope, and at which point the synchronization can be done. An alternative method to obtain a huge amount of worms is to grow them in liquid media (NGM medium without agar).
    2. Add 2 ml of alkaline hypochlorite solution (bleaching solution) to each plate and rapidly collect the worms by pipetting (about 10 sec per plate) in a sterile 15 ml conical tube. Date and label the tube with the name of the worm strain. Alternatively, harvest the worms with 1 ml of M9 buffer and collect them into 15 ml conical tubes. Centrifuge in the Eppendorf 5810 R centrifuge, using swing bucket rotor (A-4-81) with adapters for 15 ml conical tubes at 1,740 x g for 2 min at 4 °C. Discard the M9 buffer and add 2 ml of the bleaching solution. In this way the worms are washed once.
    3. Quickly start to vortex the worms for 7 min at setting 8 on the vortex and check under the stereomicroscope if the adult worms are lysed and only the eggs are present.

      Note: Steps B2 and B3 must be carried out with care as they are critical for the eggs to hatch.

    4. Without any delay, centrifuge in the Eppendorf 5810 R centrifuge at 1,740 x g for 2 min at 4 °C.
    5. Aspirate the supernatant, but leave approximately 1 ml of the solution.
    6. Adding 5 ml of M9 buffer and repeat the centrifugation and aspiration as in steps B4 and B5.
    7. Repeat 4 more times step B6, in order to remove any trace of the bleaching solution.
    8. Incubate the eggs in the final 1 ml of M9 buffer overnight at 20 °C. During this time the eggs will hatch and produce L1 stage worms.

  3. Worm under treatment
    1. Bacteria preparation
      1. In 3 different tubes, add 10 ml LB medium and 10 µl ampicillin (100 mg/ml).
      2. In the 1st tube, add a few colonies of E. coli HT115DE3/empty vector L4440.
      3. In the 2nd tube, add a few colonies of E. coli HT115DE3/pL4440-oct-1.
      4. In the 3rd tube, add a few colonies of E. coli HT115DE3/pL4440-oct-2.
      5. Incubate all the tubes for 5 h in a 37 °C orbital shaker.
    2. Drug plate preparation
      1. Melt a bottle of 500 ml of solid NGM as above (see C. elegans growth media preparation above).
      2. For normal drug plates-no knockdown
        1. Transfer 30 ml of melted NGM media kept at 55 °C into a small sterile bottle. This will make 3 Petri dishes (60 x 15 mm) each containing ~10 ml of NGM media for triplicate assays.
        2. Add the doxorubicin (stock 2 mg/ml) to the desired final concentrations for each 6 ml of NGM media and pour onto the 60 x 15 mm Petri dish. For doxorubicin use between 0.1 to 100 µM depending on the strains. Uptake was observed with as low as 0.5 µM of doxorubicin.
          IMPORTANT: Do not forget to prepare a plate without drug.
        3. Leave plates for at least 15 min at room temperature for the medium to solidify.
        4. Add 70 µl E. coli HT115DE3/pL4440-empty vector bacteria.
        5. Incubate the plates overnight at 37 °C.
      3. For knockdown drug plates
        1. Transfer 30 ml of melted NMG medium kept at 55 °C into a small sterile bottle.
        2. Add 30 µl of 1 M IPTG, needed to induce the production of dsRNA from the L4440 vector carrying a fragment of the target gene.
        3. Add to 6 ml of this media the desired concentrations of doxorubicin as above.
          IMPORTANT: Do not forget to prepare a plate without drug.
        4. Pour 6 ml of media with and without IPTG and with doxorubicin onto 60 x 15 mm Petri dishes.
        5. Dry the plates, as above.
        6. Add 70 µl E. coli HT115DE3/pL4440-oct-1 bacteria for 1st set of 5 plates.
        7. Add 70 µl E. coli HT115DE3/pL4440-oct-2 bacteria for 2nd set of 5 plates.
        8. Incubate the plates overnight at 37 °C.
    3. Now all the plates are ready to add 50 µl of the L1 synchronized worms.
    4. Verify if there are nearly 50 L1-staged worms on the plates under the stereomicroscope.
    5. Incubate the plates at 20 °C for 3 days.

  4. Microscope visualisation
    1. Preparation of slides
      1. On a frosted microscope slide add one drop (~50 µl) of 3% agarose and squeeze it with another slide to form a sandwich. After 10 sec, remove one of the slide to expose the agarose.
      2. Dry the slide with the agarose at 65 °C around 15-30 min using a Programmable Thermal Controller 96 Well.
    2. Mounting the worms
      1. Add 7 µl of 1 M levamisol onto the surface of the dried agar on the slide.
      2. To each slide, add 10-15 young adult worms from a single treatment condition by using a platinum wire to transfer the worms.
      3. Sterilize the platinum wire using a flame before transferring the next 10-15 worms from a different treatment condition onto a new slide.
      4. Add the coverslip and seal around the slide and coverslip with clear nail polish.
    3. Microscopy visualisation
      The slide containing the worm is ready to be visualised. Place the slide on the stage of an epifluorescence microscope.
      1st Adjust the objectives
      2nd Select the filters and adjust all the necessary parameters
      3rd Once everything is ready, start capturing the images
      4th Save the images
      Below is a detail description to visualize the worms using a DeltaVision microscope.
      1. Add a small drop of DeltaVision Immersion oil 1.534 onto the slide in order to use objective 40x.
      2. Place the mounted slide on the microscope by inverting it.
      3. Adjust the eyepiece filter roll to POL filter.
      4. Open the SoftWorx software . See GE Healthcare manual (GE Healthcare, 2014).
      5. Press on the microscope.
        3 windows will be opened (Figure 1).


        Figure 1. SoftWorx desktop display. The Resolve3D window includes acquisition parameters and controls for moving the stage, the Data Collection window displays images as they are acquired, and the Filter Monitor displays the filters currently selected.

      6. On the window called Resolve3D (Figure 1).
      7. Press on the gear wheel (setting)  to open Resolve3D setting window (Figure 2).


        Figure 2. Resolve3D window setting

        1. Under MISC.
        2. Select the filters.
        3. Save settings.
        4. Under FILES (Figure 3).


          Figure 3. Display of the Files tab

        5. In the Data folder field (Figure 3), enter the directory in which to save the image.
        6. In the Experiment macros folder field (Figure 3), enter the same directory.
        7. Save settings.
        8. Done.
      8. In the Resolve3D window (Figure 1), set the following parameters in Figure 4.


        Figure 4. Setting the parameters

        1. Excitation: POL.
        2. Emission: POL.
        3. % T: 32%.
        4. Exposure: 0.025.
        5. Lens: 40x.
      9. Press on the Erlenmeyer.
      10. A window called Design/Run Experiment window will be opened (Figure 5).


        Figure 5. Design and Run experiment window

        1. Under design
          1) Experimental name field: write your file name (Figure 5).
          2) Under Sectioning
              Remove the check beside the Z sectioning.
          3) Under channels (Figure 5).


          Figure 6. The Channels in the Design and Run experiment window

          Check 1st box (Figure 6)
          Under EX filter: POL
          Automatically EM filter will be modifying
          Under% T: choose 32%
          Check 2nd box (Figure 6)
          Under EX filter: FITC
          Automatically EM filter will be modified
          Under% T: choose100%
          Check 3rd box (Figure 6)
          Under EX filter: mCherry
          Automatically EM filter will be modified
          Under% T: choose 100%

        2. Under Run before starting to acquire image
          Image file name field, write the same name you wrote under the design/Experimental name (Figure 5).
        3. Now you are ready to acquire image by pressing on the start button   as shown in Figure 7. You can adjust the image by looking on the Resolve3D window which contains the stage trails in the stage View window. This way you can focus at the pharynx level.


          Figure 7. The start button in the Design and Run experiment window

    4. Result analysis
      1. Software used: ImageJ
        1. Drag the file to imageJ window.
        2. Under image → Transform → rotate the image to get an image of the worm placed anterior to posterior.
        3. Under image → Stacks → Stack to images.
        4. Under image → Color → merge channels.
          Select the image which is appropriate to the desired color.
        5. Create montage: under image → Stacks → make montage.
        6. Create the 3D picture based on the intensity.
          Take the merged image.
          Under Plugins → 3D → Interactive 3D surface plot.

Data analysis

The result is representative of a single worm from an experiment where 10 worms were placed onto the slide (Figure 8). The experiment was repeated three times with identical data. Fluorescence posterior to the pharynx is autofluorescence detected from the intestine (Figure 8). The data were quantified using ImageJ according to Papaluca and Ramotar (2016).


Figure 8. Uptake of doxorubicin in the N2 wild type animals downregulated for oct-1 that causes upregulation of OCT-2. Previously, we showed that downregulation stimulated expression of oct-2, which drives uptake of drugs into C. elegans (Papaluca and Ramotar, 2016). A. Untreated; the absence of doxorubicin is illustrated by the lack of yellow fluorescence and the green background is autofluorescence from ingestion of food by the animal. B. Treated with doxorubicin (1 µM). Doxorubicin uptake is visible as yellow due to the merge of the green autofluorescence and red fluorescence from the drug. Circles show the location of the pharynx. Scale bars = 50 µm. See Data analysis for additional details.

Notes

  1. Instead of using frozen bacteria as worm food, we typically use fresh culture of the bacteria.
  2. Always work under sterile conditions. Be on the watch for fungus (white to black like spider net) and rarely mushroom (brownish spots). If this is the case, the best solution will be to bleach the worms and collect the eggs for new larvae.

Recipes

  1. LB solution
    5 g tryptone
    2.5 g yeast extract
    5 g NaCl
    ddH2O up to 500 ml
    Add 7.5 g of agar, if making solid media plates
  2. Nematode growth media (NGM)
    1.25 g peptone
    1.5 g NaCl
    8.75 g agar
    ddH2O up to 500 ml
    Autoclave
    When the media is between 50 to 55 °C, add the following constituents before pouring the plates:
    12.5 ml of 1 M KH2PO4 pH 6
    500 µl of 1 M MgSO4
    500 µl of 1 M CaCl2
    500 µl cholesterol (stock 5 mg/ml in 95% ethanol)
    500 µl of ampicillin (stock 100 mg/ml), if using the plasmid containing bacteria HT115DE3
  3. Agar pad
    3% agarose in MilliQ water
    Melt when needed
  4. Alkaline hypochlorite solution (bleaching solution) (make fresh monthly)
    8.25 ml ddH2O
    3.75 ml 1 N NaOH
    3.00 ml Bleach (Javel)
  5. M9 buffer (1 L)
    6 g Na2HPO4
    3 g KH2PO4
    5 g NaCl
    0.25 g MgSO4·7H2O
    Sterilized by autoclaving

Acknowledgments

This work was funded by the research grant (RGPIN/202432-2012) to D.R. from the Natural Science and Engineering Research Council of Canada. A brief version of this protocol was previously described (Papaluca and Ramotar, 2016).

References

  1. Burns, A. R., Wallace, I. M., Wildenhain, J., Tyers, M., Giaever, G., Bader, G. D., Nislow, C., Cutler, S. R. and Roy, P. J. (2010). A predictive model for drug bioaccumulation and bioactivity in Caenorhabditis elegans. Nat Chem Biol 6(7): 549-557.
  2. Cesar-Razquin, A., Snijder, B., Frappier-Brinton, T., Isserlin, R., Gyimesi, G., Bai, X., Reithmeier, R. A., Hepworth, D., Hediger, M. A., Edwards, A. M. and Superti-Furga, G. (2015). A call for systematic research on solute carriers. Cell 162(3): 478-487.
  3. Cheah, I. K., Ong, R. L., Gruber, J., Yew, T. S., Ng, L. F., Chen, C. B. and Halliwell, B. (2013). Knockout of a putative ergothioneine transporter in Caenorhabditis elegans decreases lifespan and increases susceptibility to oxidative damage. Free Radic Res 47(12): 1036-1045.
  4. GE Healthcare (2014). DeltaVision Imaging System User’s Manual. PN 29087880 AB.
  5. O’Reilly, L. P., Luke, C. J., Perlmutter, D. H., Silverman, G. A. and Pak, S. C. (2014). C. elegans in high-throughput drug discovery. Adv Drug Deliv Rev 69-70: 247-253.
  6. Papaluca, A. and Ramotar, D. (2016). A novel approach using C. elegans DNA damage-induced apoptosis to characterize the dynamics of uptake transporters for therapeutic drug discoveries. Sci Rep 6: 36026.
  7. Wu, X., Fei, Y. J., Huang, W., Chancy, C., Leibach, F. H. and Ganapathy, V. (1999). Identity of the F52F12.1 gene product in Caenorhabditis elegans as an organic cation transporter. Biochim Biophys Acta 1418(1): 239-244. 

简介

秀丽隐杆线虫提供了一系列优势来调查摄取转运蛋白的作用。在这里,开发了荧光显微镜方法来监测自发荧光抗癌药物多柔比星进入C的咽部。有机阳离子转运蛋白。

背景 人类细胞具有超过450个溶质载体转运蛋白,其被认为有助于摄取几种离子,营养物质以及治疗和抗癌药物(Cesar-Razquin等人,2015)。然而,大量这些摄取转运蛋白的作用和底物是未知的。线虫是一种廉价的模型生物体,它比哺乳动物细胞提供了许多优点,可以快速研究许多高度保守的生物过程。在过去十年中,这种生物一直在几个药物发现计划中发挥作用,以鉴定新型小分子,例如,作为抗微生物剂和抑制氧化应激的小分子,尽管生物活性化合物的产量不太明显(Burns等人,2010; O'Reilly等人,,2014)。我们认为如果通过摄取转运蛋白进入动物细胞的分子更多和选择性地流入,则回收率可能更高。迄今为止,已经进行了三项研究以了解摄取转运蛋白在C中的作用。 elegans (Wu et al。,1999; Cheah等人,2013; Papaluca和Ramotar,2016)。因此,该生物体中摄取转运蛋白的功能和底物特异性的表征将有利于改进用于鉴定新生物活性分子的策略。在这里,我们概述了一种监测抗癌药物多柔比星摄入到C的咽中的方法。 elegans (Papaluca和Ramotar,2016)。多柔比星自发荧光可以通过几种广泛可用的检测系统如荧光显微镜来监测。我们注意到,从这种方法可以获得若干好处,包括通过竞争多柔比星摄取来寻找转运蛋白的新型治疗底物。

关键字:秀丽隐杆线虫, 有机阳离子吸收转运蛋白, 药物摄取, 多柔比星, 自发荧光药物, 落射荧光显微镜检查, RNAi喂养细菌

材料和试剂

  1. 培养皿60 x 15毫米(SARSTEDT,目录号:82.1194.500)
  2. 15毫升圆锥管
  3. 磨砂显微镜载玻片(尺寸:1×3";厚度:1-2mm)(UltiDent Scientific,目录号:170-7107A)
  4. 显微镜盖玻璃(尺寸:22×22#1.5)(Fisher Scientific,目录号:12-541B)
  5. 铂金线(Thomas Scientific,目录号:1233S71)
  6. 巴斯德移液器(Fisher Scientific,目录号:13-678-20C)
  7. 电子。大肠杆菌细菌HT115 DE3 与质粒pL4440-空载体
  8. 电子。具有质粒pL4440-oct-1(Ahringer's系列)的大肠杆菌细菌HT115 DE3 。序列验证了
  9. 电子。具有质粒pL4440-oct-2(Ahringer's系列)的大肠杆菌细菌HT115 DE3 。序列验证了
  10. 布里斯托尔N2(野生型)和RB1084 [Oct] 1(ok1051 )I]来自Caenorhabditis 遗传中心
  11. 氨苄青霉素(Sigma-Aldrich,目录号:A9518)
  12. 磷酸二氢钾(KH 2 PO 4)(Bio Basic,目录号:PB0445)
  13. 硫酸镁(MgSO 4)(Bio Basic,目录号:MRB0329)
  14. 氯化钙二水合物(CaCl 2)(Fisher Scientific,目录号:C79-500)
  15. 胆固醇(Sigma-Aldrich,目录号:C8503)
  16. 乙醇100%(任何公司的作品)
  17. 多柔比星(来自HôpitalMaisonneuve-Rosemont,Montreal,Canada的研究)。库存浓度为2 mg/ml
  18. IPTG(Bio Basic,目录号:PRB0447)
  19. 盐酸左旋咪唑(MP Biomedical,目录号:155228)
  20. 透明的指甲油,来自美洲马的Wild Shine
  21. 用于Luria Broth(LB)培养基的Tryptone(Bio Basic,目录号:TG217(G211))
  22. 酵母提取物(Wisent Bioproducts,目录号:800-150-LG)用于LB培养基
  23. 氯化钠(NaCl)(Wisent Bioproducts,目录号:600-082)
  24. 细菌性琼脂(Wisent Bioproducts,目录号:800-010-CG)
  25. 线虫生长培养基(NGM)的胨(Wisent Bioproducts,目录号:800-157-LG)
  26. 琼脂糖(Wisent Bioproducts,目录号:800-015-CG)
  27. 氢氧化钠(NaOH)(Bio Basic,目录号:SB0617)
  28. Bleach Lavo Pro6(Lavo Inc,Montreal,Canada)
  29. 磷酸二氢钠(Na 2 HPO 4)(Bio Basic,目录号:S0404)
  30. LB溶液(参见食谱)
  31. 线虫生长培养基(NGM)(见食谱)
  32. 琼脂垫(见食谱)
  33. 碱性次氯酸盐溶液用于漂白蠕虫(见食谱)
  34. M9缓冲区(见配方)

设备

  1. 孵化器在20°C,但范围为15至37°C(SHEL LAB,型号:2020)
  2. 37℃孵化器(Panasonic Healthcare,型号:Mir-262)
  3. 37°C振动筛(Inforst,型号:Multitron Standard)
  4. 500毫升玻璃瓶(Wheaton带有内衬帽的刻度玻璃介质瓶)
  5. 微波(变频器型号,松下医疗)
  6. 55°C水浴(Precision Scientific,目录号:66800)
  7. 金属铲(VWR)
  8. 火焰
  9. 中性培养管16 x 15 mm
  10. Pyrex锥形瓶不同大小的细菌培养
  11. 高压灭菌器
  12. 立体显微镜Leica MZ 8(Leica 10445538计划显微镜物镜1.0x)(Leica,型号:MZ 8)
  13. DeltaVision精英恢复系统(GE Healthcare,型号:DeltaVision Elite High Resolution Microscope)和 DeltaVision成像系统用户手册
  14. Fisher Vortex Genie 2(Fisher Scientific,目录号:12-812)
  15. Eppendorf 5810 R离心机(Eppendorf,型号:5810 R)
  16. VWR摇摆摇摆机基础(VWR)
  17. Ptc-100可编程热控制器96孔(Bio-Rad实验室,型号:Ptc-100 可编程热控制器)
  18. 离心机(Sigma离心机,型号:Sigma 1-14)

软件

  1. ImageJ成像软件
  2. SoftWorx软件

程序

  1. 用于蠕虫生长的细菌和培养基的制备
    1. 细菌制备(食管)
      1. 在10ml LB培养基中加入10μl氨苄青霉素(100 mg/ml)
      2. 接种一些E.的殖民地。将大肠杆菌 HT115 DE3 将载体细菌倒入LB +氨苄青霉素培养基中。
        注意:替代细菌:大肠杆菌OP50。
      3. 在旋转振荡器中以200rpm在37℃下孵育6小时
    2. ℃。线虫生长培养基制备
      1. 使用微波(功率2级25分钟)将玻璃瓶(500毫升)中的固体NGM熔化25分钟。
      2. 将熔融的介质保持在55℃,水浴15-20分钟
      3. 使用前,添加:
        12.5ml 1M KH 2 PO 4 pH 6
        500微升1M MgSO 4
        500μl1M CaCl 2
        500μl胆固醇(在95%乙醇中储存5mg/ml)
        如果使用携带L4440载体或靶基因的敲低细菌菌株HT115 DE3 ,则将500μl氨苄青霉素(储备100mg/ml)。
      4. 轻轻倒转瓶子几次以混合所有成分,然后将6ml培养基倒入60 x 15 mm培养皿中。
      5. 让它在室温下干燥约1小时
      6. 一旦干燥,使用灭菌的细菌电镀棒扩散上述步骤A1中制备的70μl细菌以形成草坪。细菌的光密度OD 600小于1.0。或者,细菌可以3滴分散在板上。
      7. 在37℃孵育板过夜。
    3. 蠕虫生长
      1. 从生长的蜗杆板上切下4块约1×1厘米的琼脂,并用100%乙醇和火焰灭菌。
      2. 将每个琼脂片转移到步骤A2中制备的4个板中的每一个上。
      3. 在20°C下孵育这些蜗杆2天。

  2. 蠕虫同步
    1. 预计4个板将填充成虫,通过立体显微镜可视化,此时可以进行同步。获得大量蠕虫的另一种方法是在液体培养基(不含琼脂的NGM培养基)中培养它们。
    2. 向每个板中加入2ml碱性次氯酸盐溶液(漂白溶液),并通过在无菌的15ml锥形管中移液(每片约10秒)快速收集蠕虫。用蠕虫菌株的名称日期并标记管。或者,用1ml M9缓冲液收获蠕虫,并将其收集到15ml锥形管中。离心机在Eppendorf 5810 R离心机中,使用带有适配器的旋转叶轮转子(A-4-81),在4°C下用1740 x g的15 ml锥形管连接2分钟。弃去M9缓冲液,加入2ml漂白溶液。以这种方式,蠕虫被洗涤一次。
    3. 迅速开始涡旋7分钟,在漩涡上设置8,并检查立体显微镜下成年蠕虫是否裂解,只有鸡蛋存在。

      注意:步骤B2和B3必须小心进行,因为它们对蛋孵化至关重要。

    4. 没有任何延迟,将离心机放在Eppendorf 5810 R离心机中,在4℃下以1,740×g离心2分钟。
    5. 吸出上清液,但留下约1ml的溶液。
    6. 加入5ml M9缓冲液,重复离心和抽吸,如步骤B4和B5。
    7. 重复4次以上步骤B6,以便去除任何痕量的漂白溶液。
    8. 在最终的1ml M9缓冲液中孵育蛋在20℃过夜。在这段时间里,鸡蛋会孵出并产生L1阶段的蠕虫

  3. 蠕虫治疗
    1. 细菌制备
      1. 在3种不同的管中,加入10ml LB培养基和10μl氨苄青霉素(100mg/ml)
      2. 在1 st 管中,添加一些殖民地的E。大肠杆菌 HT115 DE3 /空载体L4440。
      3. 在2 nd 管中,添加一些殖民地的E。大肠杆菌 HT115 DE3 /pL4440-oct-1。
      4. 在3 rd 管中,添加一些殖民地的E。大肠杆菌 HT115 DE3 /pL4440-oct-2。
      5. 将所有管子在37°C的轨道振荡器中孵育5小时。
    2. 药片制备
      1. 融化一瓶如上所述的500ml固体NGM(参见上文的生长培养基制备)。
      2. 对于正常的药物盘 - 没有击倒
        1. 将保持在55℃的30ml熔融的NGM培养基转移到小的无菌瓶中。这将产生3个培养皿(60 x 15毫米),每个培养皿含有〜10ml的NGM培养基,用于一式三份测定。
        2. 将多柔比星(库存2 mg/ml)添加到每6 ml NGM培养基的期望最终浓度,倒入60 x 15 mm培养皿。对于多柔比星使用0.1至100μM取决于菌株。观察到吸收低至0.5μM的多柔比星。
          重要:不要忘记准备没有药物的药片。
        3. 在室温下放置板至少15分钟使培养基固化。
        4. 加入70μlE。大肠杆菌HT115 DE3 /pL4440-空载体细菌。
        5. 在37℃孵育板过夜。
      3. 用于击倒药片
        1. 将保持在55℃的30ml熔融的NMG培养基转移到小的无菌瓶中
        2. 添加30μl1 M IPTG,需要从携带靶基因片段的L4440载体诱导dsRNA的产生。
        3. 加入6 ml的这种培养基中所需要的多柔比星浓度如上所述 重要:不要忘记准备没有药物的药片。
        4. 将6ml含有和不含IPTG的培养基和多柔比星倒入60 x 15 mm培养皿中。
        5. 如上所述烘干板材。
        6. 加入70μlE。大肠杆菌 HT115 DE3 /pL4440-oct-1细菌为1个<5>的5个板组。
        7. 加入70μlE。大肠杆菌 HT115 DE3 /pL4440-oct-2细菌,用于2组5个平板。
        8. 在37℃孵育板过夜。
    3. 现在所有的板都准备好添加50μl的L1同步蠕虫。
    4. 验证在立体显微镜下板上是否有近50个L1级蠕虫。
    5. 将板在20℃下孵育3天

  4. 显微镜可视化
    1. 准备幻灯片
      1. 在磨砂的显微镜载玻片上加入一滴(约50μl)的3%琼脂糖,并用另一个载玻片挤压形成三明治。 10秒后,取出一张幻灯片,露出琼脂糖。
      2. 使用可编程热控制器96孔,在65°C约15-30分钟时用琼脂糖干燥载玻片。
    2. 安装蠕虫
      1. 将7μl1 M左旋咪唑加入到载玻片上干燥琼脂的表面上
      2. 在每张幻灯片中,通过使用铂丝来传播蠕虫,从单一治疗条件中添加10-15只年轻成年蠕虫。
      3. 在将下一个10-15个蠕虫从不同的治疗条件转移到新的载玻片上之前,用火焰灭菌铂金线。
      4. 加上盖玻片,用清漆指甲油将玻璃盖和盖玻片密封。
    3. 显微镜可视化
      包含蠕虫的幻灯片已准备好可视化。将载玻片放置在落射荧光显微镜的台上。
      1 st 调整目标
      2 nd 选择过滤器并调整所有必要的参数
      3 rd 一切准备就绪,开始捕获图像
      4 th 保存图像
      以下是使用DeltaVision显微镜可视化蠕虫的详细说明。
      1. 将一滴DeltaVision浸入油1.534添加到幻灯片上,以便使用40x目标。
      2. 将安装的幻灯片倒置在显微镜上。
      3. 将目镜滤光片调整到POL滤镜。
      4. 打开SoftWorx软件。见GE Healthcare手册(GE Healthcare,2014)。
      5. 按显微镜。
        将打开3个窗口(图1)。


        图1. SoftWorx桌面显示。 Resolve3D窗口包括采集参数和用于移动舞台的控件,"数据收集"窗口将显示图像,并且"过滤器监视器"显示当前选择的过滤器。 />
      6. 在名为Resolve3D的窗口上(图1)。
      7. 按下齿轮(设定)  打开Resolve3D设置窗口(图2)。


        图2. Resolve3D窗口设置

        1. 在MISC下。
        2. 选择过滤器。
        3. 保存设置。
        4. 在文件下(图3)。


          图3.显示"文件"选项卡

        5. 在数据文件夹字段(图3)中,输入保存图像的目录。
        6. 在实验宏文件夹字段(图3)中,输入相同的目录。
        7. 保存设置。
        8. 完成。
      8. 在Resolve3D窗口(图1)中,设置图4中的以下参数。


        图4.设置参数

        1. 激励:POL。
        2. 排放:POL。
        3. %T:32%。
        4. 曝光:0.025。
        5. 镜头:40x。
      9. 按Erlenmeyer。
      10. 将打开一个名为"设计/运行实验"窗口的窗口(图5)。


        图5.设计并运行实验窗口

        1. 在设计下
          1)实验名称字段:写入您的文件名(图5)。
          2)在Section
          下     删除Z部分旁边的检查。
          3)在频道下(图5)

          图6.设计和运行实验窗口中的通道

          检查1 st 框(图6)
          在EX过滤器下:POL
          自动EM滤波器将修改
          在%T下:选择32%
          检查2 nd 框(图6)
          在EX过滤器下:FITC
          自动EM滤波器将被修改
          在%T下:选择100%
          检查3 rd 框(图6)
          在EX过滤器下:mCherry
          自动EM滤波器将被修改
          在%T下:选择100%

        2. 开始运行之前,开始获取图像
          图像文件名称字段,写下您在设计/实验名称下编写的相同名称(图5)。
        3. 现在您可以通过按开始按钮来获取图像如图7所示。您可以通过在Stage View窗口中查找包含舞台轨迹的Resolve3D窗口来调整图像。这样您就可以专注于咽部水平。


          图7."设计和运行实验"窗口中的开始按钮

    4. 结果分析
      1. 使用的软件:ImageJ
        1. 将文件拖动到imageJ窗口。
        2. 在图像下→变换→旋转图像以获得放置在后方的蠕虫的图像。
        3. 在图像下→堆叠→堆叠到图像。
        4. 图像下→彩色→合并频道。
          选择适合所需颜色的图像。
        5. 创建蒙太奇:在图像下→堆叠→进行蒙太奇。
        6. 根据强度创建3D图片。
          拍摄合并图像。
          在"插件"→"3D"→"交互式3D"曲面图。

数据分析

结果代表了一个实验的单个蠕虫,其中10个蠕虫被放置在载玻片上(图8)。用相同的数据重复实验3次。从咽后的荧光是从肠中检测到的自发荧光(图8)。使用ImageJ根据Papaluca和Ramotar(2016)量化数据。


图8.对于导致OCT-2上调的 oct-1 的N2野生型动物中的多柔比星摄取。 之前,我们显示下调刺激了Oct-2的表达,这促使药物摄入到C。 elegans (Papaluca和Ramotar,2016)。未经处理缺乏多柔比星是由缺乏黄色荧光所证明的,绿色背景是由动物摄取食物的自发荧光。 B.用多柔比星处理(1μM)。由于绿色自发荧光和来自药物的红色荧光的合并,多柔比星摄取可见黄色。圈子显示咽的位置。刻度棒=50μm。有关详细信息,请参阅数据分析。

笔记

  1. 我们通常使用细菌的新鲜培养而不是使用冷冻细菌作为蠕虫食物。
  2. 始终在无菌条件下工作。在手表上的真菌(白色到黑色像蜘蛛网),很少蘑菇(棕色斑点)。如果是这种情况,最好的解决方案是漂白蠕虫,并为新幼虫收集鸡蛋。

食谱

  1. LB解决方案
    5克胰蛋白胨
    2.5克酵母提取物
    5克NaCl
    ddH <2> O至500 ml
    加入7.5g琼脂,如果制成固体培养基板
  2. 线虫生长培养基(NGM)
    1.25克蛋白胨
    1.5克NaCl
    8.75克琼脂
    ddH <2> O至500 ml
    高压灭菌器
    当介质介于50至55°C之间时,在浇注板之前添加以下组分:
    12.5ml 1M KH 2 PO 4 pH 6
    500微升1M MgSO 4
    500μl1M CaCl 2
    500μl胆固醇(在95%乙醇中储存5mg/ml)
    如果使用含有细菌的细菌,HT115 DE3
    ,将500μl氨苄青霉素(储备100mg/ml)
  3. 琼脂垫
    3%琼脂糖在MIIIiQ水中 需要时融化
  4. 碱性次氯酸盐溶液(漂白溶液)(每月新鲜)
    8.25毫升ddH 2 O -/- 3.75 ml 1N NaOH 3.00 ml漂白剂(Javel)
  5. M9缓冲区(1 L)
    6g Na 2 HPO 4
    3g KH 2 PO 4
    5克NaCl
    0.25g MgSO 4·7H 2 O→// 通过高压消毒灭菌

致谢

这项工作由D.R.的研究资助(RGPIN/202432-2012)资助。来自加拿大自然科学与工程研究理事会。以前描述了该协议的简要版本(Papaluca和Ramotar,2016)。

参考

  1. Burns,AR,Wallace,IM,Wildenhain,J.,Tyers,M.,Giaever,G.,Bader,GD,Nislow,C.,Cutler,SR和Roy,PJ(2010)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20512140"target ="_ blank">在秀丽隐杆线虫中的药物生物累积和生物活性的预测模型。 "Nat Chem Biol"6(7):549-557。
  2. Cesar-Razquin,A.,Snijder,B.,Frappier-Brinton,T.,Isserlin,R.,Gyimesi,G.,Bai,X.,Reithmeier,RA,Hepworth,D.,Hediger,MA,Edwards,AM和Superti-Furga,G。(2015)。 A要求对溶质载体进行系统研究。 细胞 162(3):478-487。
  3. Cheah,IK,Ong,RL,Gruber,J.,Yew,TS,Ng,LF,Chen,CB and Halliwell,B。(2013)。  在秀丽隐杆线虫中敲除推定的麦角硫杆菌转运蛋白降低寿命并增加对氧化损伤的易感性。 免费Radic Res 47(12):1036-1045。
  4. GE Healthcare(2014)。  DeltaVision成像系统用户手册。 PN 29087880 AB。
  5. O'Reilly,LP,Luke,CJ,Perlmutter,DH,Silverman,GA和Pak,SC(2014)。  C。 elegans 在高通量药物发现中。 Adv Drug Deliv Rev 69-70:247-253。
  6. Papaluca,A.和Ramotar,D。(2016)。使用线虫 DNA损伤诱导的细胞凋亡来表征治疗药物发现的摄取转运蛋白的动力学的新颖方法。 6:36026。 br />
  7. Wu,X.,Fei,YJ,Huang,W.,Chancy,C.,Leibach,FH和Ganapathy,V.(1999)。作为有机阳离子转运体的秀丽隐杆线虫中的F52F12.1 基因产物的特征。 Biochim Biophys Acta 1418(1):239-244。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Amirthagunabalasingam, S., Papaluca, A., Harihar, T. and Ramotar, D. (2017). Imaging the Pharynx to Measure the Uptake of Doxorubicin in Caenorhabditis elegans. Bio-protocol 7(10): e2291. DOI: 10.21769/BioProtoc.2291.
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