• 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

        Harvesting lymphocytes from Peripheral Blood

        互联网

        672

         

        Blood Harvest

        ・        Anesthetize mice with 50ml of Xylene-Ketamine(2:1) coctail

        ・        Pin animal to a board (use distal extremities) supine

        ・        Use 70% alcohol to moisten the abdomen and lower thorax

        ・        Make an midline incision using forceps and scissors from mid abdomen to xyphoid process

        ・        Dissect to the left side of the animal and open the peritoneum , exposing the liver and diaphragm

        ・        Lift the anterior chest wall with a forcep, the heart should be apparent though the translucent diaphragm

        ・        Fill a 1cc syringe with 300 ml of heparin

        ・        Holding the syringe comfortably, with one concert motion insert the needle into the heart through the diaphragm and draw blood

        ・        Should the first attempt not succeed and pneumothorax develops, immediately open the chest cavity through a midline incision (sternotomy) and draw blood directly from the heart

        Labeling lymphocytes and separating lymphocytes from the whole blood

        ・        Add 250ml of DMEM with 10% Fetal Goat Serum(FGS) to 250ml of whole blood to block nonspecific binding

        ・        Incubate at 4°C for 10 minutes

        ・        Add 1mg of appropriate antibodies to the specimen (usually about a million cells)

        ・        Incubate at 4°C for 30 minutes

        ・        Add 2ml of cold lytic buffer solution to each specimen for 2 minutes � gently shake them

        ・        After 2 minutes fill the facs tubes with PBS

        ・        Spin at 1000rpm (450g) for 10 minutes at 4°C

        ・        Aspirate supernatant to pallet using vacuum setup

        ・        Add 2ml of lytic buffer solution for the second time for the residual RBCs and fill tubes with PBS.  Spin again for 10 minutes at 1000rpm at 4°C

        ・        Aspirate supernatant with vacuum setup to pallet

        ・        Add ? ml of PBS to the pallets and gently mix them

        ・        Specimens are ready to be read by the facs machine.  They can be stored at 4°C for at least 2 days.

         

         

        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