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Isolating Taste Buds and Taste Cells from Vallate Papillae of C57BL/6J Mice for Detecting Transmitter Secretion
从C57BL/6J小鼠轮廓乳头中分离味蕾和味觉细胞以检测递质分泌   

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Abstract

Mouse is a well-accepted model for studying taste bud function. Mice readily detect and respond to taste substances that humans consider to have sweet, bitter, salty, sour and umami taste qualities. A great deal of recent research on taste receptors is based on this species. Live mice are needed for these experiments because no alternative in vitro model incorporates all elements of taste transduction and peripheral signaling. The C57BL/6J strain was selected because these mice respond robustly to many taste stimuli and because of variety of transgenic animals, such as PLCβ2-GFP and GAD67-GFP, were derived from that strain. Prior analyses on behavior, nerve responses, cellular electrophysiology and molecular biology, all conducted on C57BL/6J mice will form a solid foundation for the proposed studies (Finger et al., 2005; Huang and Wu, 2015; Huang et al., 2007). Thus, freshly euthanized animals must be used as a source of taste buds from which we will isolate taste buds and taste cells.

Keywords: Taste bud(键芽), Ca2+ imaging(钙离子成像), Biosensor(生物传感器), Transmitter(发射机)

Materials and Reagents

  1. Fire-polished borosilicate glass micropipette (World Precision Instrument) with suction apparatus (Figure 1D)
    1. Polyethylene tubing (BD, Intramedic™, model: PE#205 )
    2. 1 ml syringe (BD)
      Caution: Pipette tip must be large enough to allow taste buds to easily pass through the opening (60~80 µm and 20~30 µm for taste buds and taste cells collection, respectively). However, a wide opening will result in a large volume of solution being drawn into the suction pipette.
  2. Sylgard dish with dissecting pins (Figure 2A)
  3. Eppendorf centrifuge tubes (1.5 ml)
  4. 35 mm culture dishes (Corning)
  5. Plastic two-way valve of syringes (Figure 1D)
  6. Cell-TAK cell and tissue adhesive (Corning) coated coverslip(s)
    Caution: Apply a tiny drop of Cell-TAK onto the center of 12-mm coverslips. Wait until the droplet is completely dry. Use ethanol (75%) followed by nanopure UV water to rinse and clean coverslips.
  7. C57BL/6J strain mice (the Jackson laboratory) (Figure 1A)
  8. 100% CO2
  9. Nanopure UV water (Thermo Fisher Scientific)
  10. Collagenase A (Roche Diagnostics, catalog number: 10103578001 )
  11. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
  12. Elastase (Worthington Biochemical Corporation, catalog number: 2292 )
  13. Trypsin inhibitor (Roche Diagnostics, catalog number: 10109886001 )
  14. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  15. HEPES (Sigma-Aldrich, catalog number: H4034 )
  16. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  17. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
  18. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  19. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6297 )
  20. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
  21. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021 )
  22. BAPTA (EMD Millipore Corporation, Calbiochem, catalog number: 196418 )
  23. Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E0396 )
  24. Enzyme mixture (see Recipes)
  25. Tyrode’s buffer (see Recipes)
  26. Ca2+/Mg2+-free Tyrode’s buffer (see Recipes)

Equipment

  1. Olympus IX73 inverted fluorescence microscope (Olympus Imaging America Inc., Olympus Optical)
  2. Stereo dissecting microscope (ZEISS, ZEISS Optical) (Figure 1C)
  3. Fiber optical illuminator (Dolan-Jenner Industrial Inc.)
  4. Compressed CO2 gas in the cylinder (Figure 1B)
  5. Plastic chamber connecting with an ample length of vinyl tubing
  6. XSE analytical balance (Mettler-Toledo International Inc.)
  7. Single-sample micro osmometer (Advanced Instruments, model: 3320 )
  8. Thermo Scientific benchtop pH meter (Thermo Fisher Scientific, Accumet™, model: XL15 )
  9. Narishige microforge for fire polishing with tungsten filament, electrically heated (NARISHIGE Group, model: MF-900 )
  10. Flaming/Brown glass micropipette puller (Sutter Instruments, model: P-1000 )
  11. Recording/Perfusion chamber (Warner Instruments, model: RC-25 ) (Figure 2B)
  12. Magnetic platform (PH4) for RC-25 recording chamber, and for mounting onto a Olympus IX73 microscope (Figure 2B)
  13. Thermolyne stirrer (Thomas Scientific, model: Nuova II )
  14. Spectrafuge 7M microcentrifuge (ReGen Lab Equipment, model: SKU: W095074 )
  15. Eppendorf 5702 centrifuge (Thermo Fisher Scientific, Fisher ScientificTM, model: Eppendorf 5702 )
  16. VWR vortex mixer for vortexing the enzyme mix (VWR International, model: MINI 230V )
  17. Gilson pipettors (200 μl and 1 ml tips) (VWR International)
  18. Sharp tweezers
  19. Microsurgical scissors

Procedure

  1. Mice are euthanized by cervical dislocation under deep CO2 (100%) anesthesia (Figure 1A-B).
  2. Obtain the tongue from animals and place in Tyrode’s buffer.
  3. Pin tongue on a Sylgard dish so that vallate papillae are easily accessible (Figure 1C).
  4. Inject ~0.3 ml of enzyme mixture in the regions near the vallate papillae (on each side) subepithelially. The needle should be visibly beneath the epithelium.
    Note: Delivering enzyme mixture by inserting the needle too deep will result in the digestion of the muscle tissue. If the location of needle is too shallow, the epithelium will be pierced, the enzyme mixture may leak through the holes on the lingual epithelium.


    Figure 1. Euthanization of mice, and excision of tougues. The photograph shows (A) the animal, C57BL/6J mouse, (B) the apparatus for euthanizing animals, (C) the tongue pinned on a Sylgard dish under the stereo dissecting microscope, and (D) the suction apparatus for collecting taste buds.

  5. Incubate the tongue in 5~10 ml of Tyrode’s buffer with aeration for 30 min.
  6. Re-pin the tongue so that vallate papillae are readily accessible (Figure 1C).
  7. Cut the epithelium surrounding vallate papillae with microsurgical scissors.
  8. Peel the epithelium away from the tongue using a sharp set of tweezers.
  9. Pin the pieces of epithelium, serous side up, onto a Sylgard dish. Stretch the epithelium so that each papilla is exposed and easily accessible (Figure 2A).
  10. Exchange the solution for Ca2+/Mg2+-free Tyrode’s buffer. Incubate the epithelium in the buffer for 20 min for collecting individual taste buds or 30 min for single taste cells.
  11. Replace Ca2+/Mg2+-free Tyrode’s buffer in the Sylgard dish with regular Tyrode’s buffer.
  12. Using a fire-polished glass micropipette (60~80 μm) (Figure 1D), sweep each papilla several times while applying suction. Periodically stop and discharge the solution onto a Cell-TAK treated coverslip.
    Critical: There are, typically, sufficient number of taste buds on the vallate papillae to make 2~4 coverslips.


    Figure 2. Suction apparatus setup to collect taste buds from mouse vallate papillae. A. The photo shows the peeled lingual epithelium, serous side up, pinned on a Sylgard dish. B. Isolation of taste buds is performed in the Sylgard dish, and then taste buds are delivered via a glass micropipette to a recording chamber assembled on the platform. C. A Nomarski optics micrographic image of isolated taste buds in a living preparation when viewed under the Olympus IX73 microscope.

  13. For collecting single cells, gentle trituration (suck Tyrode’s containing taste buds into the glass pipette and discharge it immediately for repeating 5-6 times) is required via 20~30 μm fire-polished glass pipettes.
    Note: Validating the intact membranes can be a criterion for healthy-looking taste buds/cells. Taste cells may be not healthy if the intracellular Ca2+ concentration is over 200 nM at their resting stage (Critical!).
  14. Allow 20 min for taste buds and/or taste cells to settle, and to attach to the bottom of the chamber, which is made by glass coverslips (Figure 2B-C).

Recipes

  1. Enzyme mixture
    Collagenase A 1 mg/ml
    Dispase II 2.5 mg/ml
    Elastase 1 mg/ml
    Trypsin inhibitor 1 mg/ml
  2. Tyrode’s buffer
    310-320 Osm and applied at pH 7.2
    CaCl2 2 mM
    HEPES 10 mM
    KCl 5 mM
    MgCl2 1 mM
    NaCl 140 mM
    NaHCO3 10 mM
    Na-pyruvate 10 mM
    Glucose 10 mM
  3. Ca2+/Mg2+-free tyrode’s buffer (pH 7.2)
    CaCl2 0 mM
    HEPES 10 mM
    KCl 5 mM
    MgCl2 0 mM
    NaCl 140 mM
    NaHCO3 10 mM
    Na-pyruvate 10 mM
    Glucose 10 mM
    BAPTA 2 mM
    EGTA 2 mM

Acknowledgments

This work was supported by SIUSOM research seed grant (AYH).

References

  1. Finger, T. E., Danilova, V., Barrows, J., Bartel, D. L., Vigers, A. J., Stone, L., Hellekant, G. and Kinnamon, S. C. (2005). ATP signaling is crucial for communication from taste buds to gustatory nerves. Science 310(5753): 1495-1499.
  2. Huang, A. Y. and Wu, S. Y. (2015). Calcitonin gene-related peptide reduces taste-evoked ATP secretion from mouse taste buds. J Neurosci 35(37): 12714-12724.
  3. Huang, Y. J., Maruyama, Y., Dvoryanchikov, G., Pereira, E., Chaudhari, N. and Roper, S. D. (2007). The role of pannexin 1 hemichannels in ATP release and cell-cell communication in mouse taste buds. Proc Natl Acad Sci U S A 104(15): 6436-6441.

简介

鼠标是研究味蕾功能的一个被广泛接受的模式。小鼠容易检测和响应人们认为具有甜味,苦味,咸味,酸味和鲜味的品质的味道。最近对味觉感受器的大量研究是基于这个物种。这些实验需要活的小鼠,因为没有替代的体外模型包含所有的味觉转导和外周信号的元件。选择C57BL / 6J菌株,因为这些小鼠对许多味觉刺激反应强烈,并且由于来自该菌株的多种转基因动物,例如PLCβ2-GFP和GAD67-GFP。对C57BL / 6J小鼠进行的行为,神经反应,细胞电生理学和分子生物学的先前分析将为拟议研究奠定坚实的基础(Finger et al。,2005; Huang and Wu,2015; Huang et al。,2007 )。因此,新鲜安乐死的动物必须用作味蕾的来源,我们将从中分离出味蕾和味道细胞。

关键字:键芽, 钙离子成像, 生物传感器, 发射机

材料和试剂

  1. 火抛光的硼硅酸盐玻璃微量吸移管(世界精密仪器)带有吸气装置(图1D)
    1. 聚乙烯管(BD,Intramedic TM,型号:PE#205)
    2. 1 ml注射器(BD)
      注意:移液管尖端必须足够大,以允许味蕾轻松   通过开口(60〜80μm和20〜30μm为味蕾和 味细胞收集)。 然而,一个大开口将 导致大体积的溶液被吸入抽吸 移液器。
  2. 具有解剖针的Sylgard皿(图2A)
  3. Eppendorf离心管(1.5ml)
  4. 35mm培养皿(Corning)
  5. 注射器的塑料二通阀(图1D)
  6. 细胞-TAK细胞和组织粘合剂(Corning)涂布的盖玻片
    注意:在12-mm盖玻片的中心涂抹一滴Cell-TAK。 等待液滴完全干燥。 使用乙醇(75%),然后用纳米UV水冲洗和清洁盖玻片。
  7. C57BL/6J株小鼠(Jackson实验室)(图1A)
  8. 100%CO 2
  9. Nanopure UV水(Thermo Fisher Scientific)
  10. 胶原酶A(Roche Diagnostics,目录号:10103578001)
  11. Dispase II(Roche Diagnostics,目录号:04942078001)
  12. 弹性蛋白酶(Worthington Biochemical Corporation,目录号:2292)
  13. 胰蛋白酶抑制剂(Roche Diagnostics,目录号:10109886001)
  14. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  15. HEPES(Sigma-Aldrich,目录号:H4034)
  16. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  17. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M4880)
  18. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  19. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S6297)
  20. 丙酮酸钠(Sigma-Aldrich,目录号:P5280)
  21. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G7021)
  22. BAPTA(EMD Millipore Corporation,Calbiochem,目录号:196418)
  23. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E0396)
  24. 酶混合物(见配方)
  25. Tyrode的缓冲区(参见配方)
  26. Ca 2 + /Mg 2 + - 无Tyrode缓冲液(参见配方)

设备

  1. Olympus IX73倒置荧光显微镜(Olympus Imaging America Inc.,Olympus Optical)
  2. 立体解剖显微镜(ZEISS,蔡司光学)(图1C)
  3. 光纤照明器(Dolan-Jenner Industrial Inc.)
  4. 在气缸中压缩CO 2气体(图1B)
  5. 塑料室与大长度的乙烯管材连接
  6. XSE分析天平(Mettler-Toledo International Inc.)
  7. 单样本微量渗透压计(Advanced Instruments,型号:3320)
  8. Thermo Scientific台式pH计(Thermo Fisher Scientific,Accumet TM,型号:XL15)
  9. Narishige微型锻造钨丝灯丝,电加热(NARISHIGE集团,型号:MF-900)
  10. 火焰/棕色玻璃微量移液器(Sutter Instruments,型号:P-1000)
  11. 记录/灌注室(Warner Instruments,型号:RC-25)(图2B)
  12. 用于RC-25记录室并安装在Olympus IX73显微镜(图2B)上的磁性平台(PH4)
  13. Thermolyne搅拌器(Thomas Scientific,型号:Nuova II)
  14. Spectrafuge 7M微量离心机(ReGen Lab Equipment,型号:SKU:W095074)
  15. Eppendorf 5702离心机(Thermo Fisher Scientific,Fisher Scientific ,型号:Eppendorf 5702)
  16. VWR涡旋混合器(VWR International,型号:MINI 230V)
  17. Gilson移液器(200μl和1ml吸头)(VWR International)
  18. 镊子
  19. 显微外科剪刀

程序

  1. 在深CO 2(100%)麻醉下通过颈脱位将小鼠安乐死(图1A-B)。
  2. 从动物获得舌头,并放置在Tyrode的缓冲液中
  3. 将舌头放在Sylgard盘上,以便可以容易地接近vallate乳头(图1C)
  4. 注射约0.3毫升酶混合物在附近乳头乳头(在每一侧)上下区域。针应明显在上皮下。
    注意:通过插入针太深来输送酶混合物将导致肌肉组织的消化。如果针的位置太浅,上皮将被刺穿,酶混合物可能通过舌上皮上的孔泄漏。


    图1.小鼠的安乐死和损伤的切除。照片显示(A)动物C57BL/6J小鼠,(B)用于安乐死动物的装置,(C)舌头固定在Sylgard皿在立体解剖显微镜下,(D)用于收集味蕾的抽吸装置
  5. 在5〜10ml Tyrode缓冲液中通气30分钟,孵育舌头。
  6. 重新固定舌头,以便可以容易地接触到乳头乳头(图1C)
  7. 用显微外科剪刀剪切围绕vallate乳头的上皮。
  8. 使用尖锐的镊子将上皮从舌头剥离。
  9. 把上皮片,浆液一面朝上,塞到一个Sylgard菜。拉伸上皮,使每个乳头暴露和容易获得(图2A)。
  10. 交换Ca 2 + /Mg 2 + -free Tyrode's缓冲液的溶液。孵育上皮在缓冲液中20分钟收集个别味蕾或30分钟为单味细胞。
  11. 用规则Tyrode缓冲液替换Sylgard培养皿中的Ca 2 + /Mg 2 + - 无Tyrode缓冲液。
  12. 使用火抛光的玻璃微量移液管(60〜80μm)(图1D),抽吸每个乳头几次。定期停止并将溶液排放到Cell-TAK处理的盖玻片上 < em>关键:通常,在vallate乳头上有足够数量的味蕾来制备2〜4盖玻片。


    图2.显示剥离的舌侧上皮,浆液侧朝上,固定在Sylgard盘上。图2.吸引装置设置以收集来自小鼠vallate乳头的味蕾。 B.进行味蕾的分离Sylgard培养皿,然后将味蕾通过玻璃微量移液管递送到装配在平台上的记录室。 C.当在奥林巴斯IX73显微镜下观察时,活体制剂中孤立的味蕾的Nomarski光学显微图像。

  13. 为了收集单细胞,需要通过20〜30μm的火抛光玻璃移液管进行温和研磨(将含有味蕾的蒂罗德吸入玻璃移液管并立即将其排出,重复5-6次)。 注意:验证完整的膜可以是健康看起来的味蕾/细胞的标准。 如果细胞内Ca 2+浓度在其静止阶段超过200nM,则味觉细胞可能不健康(危急!)。
  14. 让味蕾和/或味细胞沉淀20分钟,并附着在由玻璃盖玻片制成的室底部(图2B-C)。

食谱

  1. 酶混合物
    胶原酶A 1mg/ml
    Dispase II 2.5mg/ml
    弹性蛋白酶1mg/ml
    胰蛋白酶抑制剂1mg/ml
  2. Tyrode的缓冲区
    310-320 Osm并在pH 7.2下施用 CaCl 2 2mM
    HEPES 10mM
    KCl 5mM
    MgCl 2 1mM
    NaCl 140mM
    NaHCO 3 3mM,
    丙酮酸钠10mM
    葡萄糖10 mM
  3. Ca 2+ +/+/Mg 2+ +无缓冲液(pH 7.2)。
    CaCl 2 <0mM,
    HEPES 10mM
    KCl 5mM
    MgCl 2 <0mM,
    NaCl 140mM
    NaHCO 3 3mM,
    丙酮酸钠10mM
    葡萄糖10 mM
    BAPTA 2mM
    EGTA 2mM

致谢

这项工作是由SIUSOM研究种子授权(AYH)支持。

参考文献

  1. Finger,T.E.,Danilova,V.,Barrows,J.,Bartel,D.L.,Vigers,A.J.,Stone,L.,Hellekant,G.and Kinnamon,S.C。(2005)。 ATP信号对于从味蕾到味觉神经的交流至关重要。科学 310(5753):1495-1499。
  2. Huang,A.Y。和Wu,S.Y。(2015)。 降钙素基因相关肽降低小鼠味蕾的味觉诱发ATP分泌。 < em> J Neurosci 35(37):12714-12724。
  3. Huang,Y.J.,Maruyama,Y.,Dvoryanchikov,G.,Pereira,E.,Chaudhari,N。和Roper,S.D。(2007)。 pannexin 1半通道在小鼠味蕾中ATP释放和细胞 - 细胞通讯中的作用。 Proc Natl Acad Sci USA 104(15):6436-6441。
  • English
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huang, A. Y. and Wu, S. Y. (2016). Isolating Taste Buds and Taste Cells from Vallate Papillae of C57BL/6J Mice for Detecting Transmitter Secretion. Bio-protocol 6(11): e1824. DOI: 10.21769/BioProtoc.1824.
  2. Huang, A. Y. and Wu, S. Y. (2015). Calcitonin gene-related peptide reduces taste-evoked ATP secretion from mouse taste buds. J Neurosci 35(37): 12714-12724.
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