Isolating Taste Buds and Taste Cells from Vallate Papillae of C57BL/6J Mice for Detecting Transmitter Secretion

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 an 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 Scientific^TM, 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