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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