• 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

        T and B cell Isolation

        互联网

        1059

         

        Reagents
        Heparin - 1000 U/ml
        Ficoll-Hypaque
        PBS
        RPMI-1640 supplemented with 10 mM glutamine and 15% FBS
        AET (0.14M) Dissolve 1.967 g AET in 35 ml di-H2O.
        Adjust to pH 8.0 with 1.0N NaOH. Bring volume to 50 ml with di-H2O.
        Store at 2-8oC. Check pH every 2 weeks.
        AET-treated SRBC
        Wash SRBC 4 times with PBS 
        Add 4 volumes AET to 1 volume packed SRBC in a 15 m conical tube (1 ml of AET + 0.25 ml packed SRBC). 
        Mix well. Incubate in a 37oC water bath for 30 minutes. Shake vigorously. 
        Wash 3 times with PBS. 
        Store in PBS at 2-8oC for up to 3 days.
        SRBC-Absorbed FBS
        Mix 10 volumes of FBS with 1 volume packed SRBC. 
        Incubate at 37oC fir 30 minutes. 
        Incubate at 2-8oC for 30 minutes. 
        Centrifuge at 400 g for 10 minutes. 
        Collect the FBS. Filter sterilize. Store aliquots at -20oC.
        Preparation of PBL's
        Draw peripheral blood into syringe containing 10 U/ml heparin.
        Dilute the blood 1:1 with PBS.
        Layer 30 ml of diluted blood onto 20 ml Ficoll-Hypaque. 
        Centrifuge at 1550 rpm for 30 minutes, room temperature. 
        Aspirate and discard the supernatant. 
        Carefully collect the interface of PBL's and transfer into a clean tube. 
        Fill the tube with PBS. Centrifuge at 1550 rpm for 10 minutes. 
        Wash the pellet 2 times with PBS. 
        Count the cells and resuspend to 107 cells/ml in PBS.
        Separation of T-Cells
        Mix 1 ml of AET-treated SRBC with 10 ml FBS.
        Mix and equal volume of PBL's with a 1% (v/v) mixture of AET-SRBC_FBS in a 50 ml tube.
        Incubate in a 37oC water bath for 10 minutes. 
        Centrifuge at 200 g for 10 minutes. Make sure that the cells have pelleted. If not, re-centrifuge for 5 minutes. 
        Place the tube upright on ice for 60 min. 
        Layer super over 15 ml of Ficoll-Hypaque leaving 7.5 ml of fluid above the pellet. 
        Resuspend the pellet by rotating the tube along the long axis. 
        Stand upright for 1 minute. Remove the top 5 ml and layer on Ficoll-Hypaque. 
        Rotate as above and transfer to gradient tube. 
        Wash the tube with 5 ml of PBS and add to gradient. 
        Centrifuge at 300 g for 40 minutes, room temperature. 
        Collect the B cells at the interface. Wash 3 times with PBS. 
        Suspend the SRBC-T cell pellet. Centrifuge at 300 d for 10 minutes. 
        Aspirate all of the supe. Break up the cell pellet by gently shaking. 
        Add 9 ml of di-H2O. with shaking for 4 seconds. 
        Add 1 ml of 10X PBS with shaking. 
        Immediately fill the tube with 1X PBS. 
        Centrifuge at 300 g for 10 minutes, and wash 2 times with PBS.

         

        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