Activation of mouse macrophages
Activation of mouse macrophages
Macrophage activation goes through two phases: a sensitization phase and an activation phase. While most protocols for macrophage activation use a one-step approach (sensitization and activation), this protocol makes a clear distinction between these two phases of macrophage activation. After a period of sensitization (48-72 h), macrophages can be examined for killing of pathogens (e.g., parasites or bacteria; Elements 6.5 and 6.6) or for synthesis of reactive nitrogen intermediates (RNI; Elements 6.5).
Author: J.E. Colligan et al, Translator: Xuitao Cao et al. This experiment is from the "Compendium of Immunology Laboratory Guidelines".
Operation method
Activation of mouse macrophages Move Basic scheme Activation of mouse macrophages Materials Mouse macrophages, resting state (Unit 6.1) DMEM-10: DMEM containing 10 % (V/V) FBS (HyCloxie) and 50 ug/1111 Gentamicin Sulphate Culture base (G I B C O /B R L ) Inflammatory macrophages: mouse peritoneal macrophages after stimulation with a proinflammatory agent (Unit 6.1) 2 4-well flat-bottomed cell culture plates (Costar) 1 . To set up the experimental and control groups (see step 1 of the basic protocol), add 0.5 m l of cell suspension per well to the 2 4-well plate. 2 . Incubate in a 37°C, 5 % C 〇 2 cell culture incubator for I h to allow the macrophages to adhere to the wall. 3 . After discarding the supernatant, slowly add Iml D E M E M - I O medium along the walls of the wells to avoid damaging the adherent cell layer. Repeat the washing process twice and add 0.5 ml of DEMEM-IO medium at the end. 4 . For sensitization and activation of macrophages, see steps 2 and 4 of the basic protocol. For more product details, please visit Aladdin Scientific website.![含 500U/ml 小鼠重组 7-干 扰 素 （IFN-y ， Pharmingen) 的 D M E M -10 E .c o G L P S (Sigma)，浓度 5iug/m l ， D M E M -10 配制 12m m X 75m m 聚丙烯塑料试管，带 盖 （Falcon) Sorvall R T 6000 4°C离心机及H 1000B 转 子 （或替代品） 1 . 用 D M E M -I O 调整小鼠腹腔巨噬细胞浓度至I X l O 6个细胞/m l 。如下所示，准 备 6 只 12m m X 75m m 试管，每管分别加入0.5m l 细胞悬液。 未刺激组 致敏组 IFN-y激活的致敏组 L P S 激活的致敏组 IFN-7激活组 L P S 激活组 2 . 加 入 ImI浓 度 为 500U / m l 的 IFN-7 (终 浓 度 2U /ml) ，置 37°C ， 5 % C 0 2细胞培养箱 中培养4h ，用以致敏巨噬细胞。 实验室可摸索各自的技术方法，用以致敏巨噬细胞。例 如 ，用不同浓度的IFN- 7 (0.1〜l〇U /ml) 刺激巨噬细胞来测定剂量反应曲线。 3 . 加 入 1： 111〇]\^]\4-10,4°(：， 40(^离心1〇 111丨11，弃上清。重 复 洗 涤 细 胞 2次 后 ，加入 0. 5ml D M E M -I O 培养基重悬细胞。 4 . 在相应试管中加入5 4 浓 度 为 500U / m l 的 IFN-7 (终 浓 度 lO U /ml) 或 ImI 浓度为 5/xg/ml的 LPS (终 浓 度 10ng/ml)。置 37°C ， 5 % C 0 2细胞培养箱中培养lh，用以激 活巨噬细胞。 用不同浓度的IFN-7 (1〜50U /ml) 或 LPS (100pg/m l 〜100ng/ml) 测定巨嗤 细胞对IFN-y 的剂量反应曲线。胞内、外的多种因子都能作为已致敏巨噬细胞的激 活信号，这些分子均可替代IFN-7。 5 . 加入111110矹£“ -10,4°(：， 40(^离心1〇 111丨11，弃上清。重 复 洗 涤 细 胞 2次 后 ，将细 胞重悬于〇• 5ml D M E M -I O 中备用](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/07/A1469600968779qp2xpmbgbepng_small.jpg)