Aging Aging

256 Taylor et al.harvested. The diameter of the micropipet is determined using a light microscopefitted with a graticule. Crude micropipets for muscle fiber work can be preparedby heating and pulling glass capillaries in a Bunsen burner flame. These micropipetscan then be heat sterilized. Prior to using these crude micropipets the tip isgently broken against the microscope slide to yield a tip of appropriate diameter(see Note 6).3. Dissection of single muscle fibers: Single muscle fibers can be picked from appropriatelystained sections (30 µm) by hand, using a standard inverted microscope.The muscle section is kept under 50% alcohol during this procedure. The micropipetis held by hand, and the tip is used to tease around the edge of an individualmuscle fiber and pick up the fiber. The presence of a cell on the capillary is determinedvisually with a hand held eyepiece.4. Dissection of neuronal cells: An inverted microscope with long-distance workingobjective lenses is used in conjunction with a micromanipulator. The micropipetsare held in position with an injection holder attached to an instrument holder onthe micromanipulator. Stained sections (30 µm) of the neuronal tissue under investigationare moistened prior to micromanipulation with TE buffer. Single neuronsare then picked up on the end of a micropipet. Visual confirmation of the presenceof a cell is obtained by focusing on the micropipet while it is still held in theinjection holder (Fig. 3) (see Note 7)5. Cell lysis: The tip of the micropipet containing the single cell is broken off intosterile microfuge tubes, containing 20 µL of TE buffer. At this point the cells canbe stored at 4°C for several weeks, prior to lysis. The microfuge tubes containingthe individual cells are centrifuged at 7000g for 10 min. The supernatant isremoved and the cells are lysed with 10 µL of 50 mM Tris-HCl, pH 8.5; 1 mMEDTA, pH 8.0; 0.5% Tween-20, and 200 µg/mL of proteinase K (33). The cellsare incubated for 2 h at 55°C, with agitation every 30 min. This is followed byheat inactivation of the proteinase K at 95°C for 10 min (see Note 8).6. PCR amplification of single cells: PCR amplification of a rarely deleted region ofmtDNA (ND1 and 16S rRNA genes) is performed to demonstrate the presence oftemplate DNA (wtDNA) within the single cell lysate. The primers are L3108 (nt3108–3127) and H3717 (nt 3717–3701) which amplify a 610-basepair product.The common deletion (mtDNA 4977 ) is amplified using the primers L8282 (nt8282–8305) and H13851 (nt 13851–13832) which yield a 593-basepair product.The cell lysate is used as the template in a single PCR reaction. A standard PCRprocedure is performed using AmpliTaq ® (1 U/reaction), PCR buffer, 0.6 µM forwardand reverse primer, and 200 µM dNTPs in a 50-µL reaction volume. PCRamplification is performed under the following conditions: initial denaturation at94°C for 2 min, denaturation at 94°C for 45 s, annealing at 51°C (wtDNA) or56°C (mtDNA 4977 ) for 45 s, and extension at 72°C for 1 min for 34 cycles followedby a final extension at 72°C for 8 min (7). PCR products (40 µL) areelectrophoresed through a 1.5% agarose gel in 1× TAE buffer containing ethidiumbromide, and visualized by UV transillumination.