[方法] isolation murine primary macrophage
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
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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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