作者changyaowen (研究生活)
看板Biotech
标题Re: [方法] isolation murine primary macrophage
时间Mon Jan 28 10:19:23 2008
※ 引述《changyaowen (研究生活)》之铭言：
哇~~~~没人做过喔
有板友要分享一下的吗
: 请问有板友做过这类的分离实验ㄇ
: 一起来讨论一下
: 我的目的 分离出mouse的peritoneal macrophage
: 然後将primary cell immortalization 变成cell line
: 我看了几遍红皮书(current protocol)的做法
: 打入TG 等7~10天 用medium去冲洗腹腔中的细胞 离心 culture
: isolation遇到的问题
: 1. 在冲洗的过程 常常会去吸到组织或脂肪 非常不容易吸出medium
: 2. 只要针一出体外 要再进去基本上很会漏medium
: 3. 洗的过程 有时会不小心去吸到血液 应当是碰到血管了 有好的避免方法ㄇ
: 4. 外观可看出腹腔中还有不少的medium 只是吸不出来
: 其他就是 culture过程 有板友有照过图可以看ㄇ
: 还是用怎样的方法去assay该cell是你要的macrophage
: 一起讨论一下罢
: ============================================================================
: 基本上 按照以下节录红皮书的方法:
: Harvest peritoneal cells
: 1a. To collect resident peritoneal cells: Euthanize untreated mice by decapitation or CO2 asphyxiation.
: 1b. To collect inflammatory macrophages: Fill 6-ml syringe with 1.0 ml of 3% proteose peptone. Attach 25-G needle and inject
: solution into peritoneal cavity of each mouse. Allow inflammatory response to proceed for 3 days and euthanize by
: decapitation or CO2 asphyxiation. Alternatively, inject 1.0 ml of 3% Brewer thioglycollate medium into peritoneum 5 to 7
: days prior to cell harvest (this will yield a larger number of inflammatory macrophages).
: Use only mice bred and housed in clean environments (preferably barrier facilities, UNIT 1.2). Chronic, endemic
: infectious diseases, such as those caused by Sendai virus and mouse hepatitis virus, have a profound effect on macrophage
: physiology and will affect cell responsiveness and capacity for function.
: Decapitation or CO2 asphyxiation are the preferred methods of euthanasia for macrophage isolation because they limit the
: chance of contaminating the peritoneal cavity with blood.
: 2. Wet the abdomen of each mouse with 70% alcohol to sterilize the area.
: 3. Make a midline incision with sterile scissors. Retract abdominal skin with forceps to expose the intact peritoneal
: wall.
: 4. Attach 30-cc syringe to 19-G needle and fill with harvest medium. Push on syringe plunger to allow a small amount of
: medium to pass through the needle as the needle penetrates the peritoneum to avoid hitting the intestines. With
: bevelled end of needle facing up, insert needle through peritoneal wall at the midline. Inject 10 ml harvest medium
: into each mouse. Inject and collect fluid from three mice using a single 30-cc syringe. Passing a small amount
: of medium through the needle serves to eliminate any air bubbles.
: 5. Using the same syringe and needle, insert needle bevelled end down into peritoneum. Raise needle slightly to cause
: tenting of peritoneal wall. Withdraw peritoneal fluid slowly.Expect fluid recovery of ~ 8 ml/mouse.
: 6. Remove needle from syringe and dispense pooled peritoneal fluid to 50-ml
: polypropylene centrifuge tubes on ice.
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