• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Isolation of Mitochondria

        互联网

        855
         

        Materials

         

        • Rat, mouse or suitable source of fresh liver
        • 0.25 M sucrose in 10 mM HEPES buffer, pH 7.5 (Homogenization buffer)
        • 0.25 M sucrose in 10 mM HEPES, pH 7.5 + 1 mM EDTA (Suspension buffer)
        • Teflon homogenizer
        • Refrigerated centrifuge
        • Janus Green B
        • Hemacytometer and microscope

        Procedure

         

        1. Sacrifice and exsanquinate a rat that has not been fed for at least 24 hours prior to lab.

           

        2. Remove the liver and weigh it.

           

        3. Add the liver to a beaker, and for each gram of liver, add 9.0 ml of 0.25 M sucrose in 10 mM HEPES buffer, pH 7.5. This will produce a 10% brei, a term used to indicate a homogenized suspension.

           

        4. Add the brei to centrifuge tubes and centrifuge at 4,500 xg for 10 minutes at 4° C.

           

        5. Decant the supernatant into clean centrifuge tubes and discard the pellet.

           

        6. Recentrifuge the supernatant at 16,000 xg for 25 minutes at 4° C.

           

        7. Decant and discard the supernatant. Resuspend the pelleted mitochondria in 20 ml of 0.25 M sucrose in 10 mM HEPES. Skip to step 10.

          OR

          Optional: if cleaner mitochondria are desired, resuspend in 20 ml of 0.25 M sucrose in 10 mM HEPES + 1 mM EDTA and perform steps 8 and 9.

           

        8. Recentrifuge the suspended pellet at 16,000 xg for 25 minutes at 4° C.

           

        9. Decant and discard the supernatant. Resuspend the washed pellet in 20 ml of fresh sucrose without EDTA and place the suspension in an ice bath until further use is required. The suspension will remain active for approximately 4-6 hours if kept cold.

           

        10. Mix a few drops of Janus Green B solution with 0.1 ml of mitochondrial suspension. Place one drop of this mixture in a hemocytometer and determine the number of mitochondria per ml. If there are too many mitochondria to count, make serial dilutions of 1/10 to 1/1000 and recount. Diluted mitochondria must be counted rapidly. They are not stable and will decompose if not counted within a few minutes of the dilution.

           

          <center> <font>Record the number of mitochondria/ml______________ </font></center>
        <center> <p>  </p> </center>
        上一篇:The Hill Reaction   下一篇:DAPI染色
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序