2 Materials

1. Dulbecco’s minimum essential medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin plus streptomycin (DMEM+).
2. Phosphate-buffered saline (PBS).
3. PBS containing 2 mM EDTA.
4. Polypropylene, 50-ml sterile, conical centrifuge tubes (Becton Dickinson Labware).
5. Polystyrene, 5.5-ml tubes (Becton Dickinson Labware).
6. 6-well plate.
7. Fluorescent-labelled Fluoresbrite microspheres, 1 μm diameter(Polysciences, Inc., Warrington, PA).
8. Trypan blue solution.
9. Propidium iodide solution 1 mg/ml.
10. Flow cytometer.
11. Incubator.

3 Methods

1. Microglia isolation is described elsewhere in this volume. The procedure, how microglia are isolated, is not relevant to the phagocytosis assay.
2. Centrifuge isolated microglia at 475 × g for 6 min and discard the supernatant.
3. Resuspend the pellet in fresh DMEM+ and determine the viable cell number by trypan blue staining.
4. Adjust the cell density to 2.5 × 10 5 cells per ml. Add 2 ml of cell suspension (i.e., 5 × 10 5 cells) to each well of a 6-well plate and incubate for 2 h at 37 °C in a 5 % CO 2 humidifi ed incubator. This procedure allows the viable microglia to adhere to the cell
   3.1 Plating and Preparation of Microglia Phagocytosis Assay for Microglia culture plastic while the dead cells remain fl oating in the supernatant.
5. Aspirate the supernatants with floating cell debris and add 2 ml fresh DMEM+ to each well. Return the plates to the incubator for another 12–16 h.

译文

2 .材料

1. 杜尔贝科最低必需培养基添加10%胎牛血清(FBS)和1%青霉素+链霉素(DMEM+)。
2. 磷酸盐缓冲盐水(PBS)。
   3.含有2毫米EDTA的PBS。
3. 聚丙烯，50ml无菌锥形离心管(Becton Dickinson Labware)。
4. 聚苯乙烯，5.5毫升试管(Becton Dickinson Labware)。
5. 6-well盘子。7. 荧光标记荧光石微球，直径1 μm (polyciences, Inc.， Warrington, PA)。
6. 台盼蓝溶液。
7. 碘化丙啶溶液1mg /ml。10. 流式细胞分析仪。
8. 孵化器。

3.方法

1.小胶质细胞分离在本卷的其他地方描述。小胶质细胞如何分离的过程与吞噬实验无关。
2. 分离出的小胶质细胞以475 × g离心6分钟，弃去上清液。
3.将颗粒重悬于新鲜DMEM+中，台盼蓝染色测定活细胞数。
4.将细胞密度调整为每毫升2.5 × 10 5个细胞。在6孔板的每孔中加入2毫升细胞悬液(即5 × 10 5个细胞)，在5% co2加湿培养箱中37℃孵育2小时。3.1 小胶质细胞吞噬实验的制备小胶质细胞培养塑料，而死细胞仍漂浮在上清液中。
5. 用漂浮细胞碎片抽吸上清液，每孔加入2毫升新鲜DMEM+。将培养皿放回培养箱再培养12-16小时。