作者changyaowen (研究生活)
看板Biotech
標題Re: [方法] isolation murine primary macrophage
時間Mon Jan 28 10:19:23 2008
※ 引述《changyaowen (研究生活)》之銘言：
哇~~~~沒人做過喔
有板友要分享一下的嗎
: 請問有板友做過這類的分離實驗ㄇ
: 一起來討論一下
: 我的目的 分離出mouse的peritoneal macrophage
: 然後將primary cell immortalization 變成cell line
: 我看了幾遍紅皮書(current protocol)的做法
: 打入TG 等7~10天 用medium去沖洗腹腔中的細胞 離心 culture
: isolation遇到的問題
: 1. 在沖洗的過程 常常會去吸到組織或脂肪 非常不容易吸出medium
: 2. 只要針一出體外 要再進去基本上很會漏medium
: 3. 洗的過程 有時會不小心去吸到血液 應當是碰到血管了 有好的避免方法ㄇ
: 4. 外觀可看出腹腔中還有不少的medium 只是吸不出來
: 其他就是 culture過程 有板友有照過圖可以看ㄇ
: 還是用怎樣的方法去assay該cell是你要的macrophage
: 一起討論一下罷
: ============================================================================
: 基本上 按照以下節錄紅皮書的方法:
: 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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