荧光微球吞噬实验的Protocol (from dxy)

dr.qinan

最近有两位战友发信询问我的荧光微球吞噬实验的Protocol。

现分享于此：

Latex Beads Phagocytosis assay

---Materials

*Fluorescence labeled latex beads (1um diameter), 2.5% aqueous suspension
* 3% BSA containing 25 mM Na2HPO4, pH 6.0
* 0,3%(w/v) azide
* Culture medium containing 5% FBS
* Distilled water, PBS
* Bath sonication, 6(12)-well plates

---Cell culture and treatment

1, Inoculate plates with 7,0104 cells/cm2 per well. Incubate at 37℃,5% CO2 for 24 hr, best until 50-70% confluence is reached

2, Remove culture medium and expose the cells to test material. Incubate at 37℃,5% CO2 for 24 hr.

---Preparation of coated latex beads

1， Wash latex beads with distilled water and pellet at 10,000g for 8 min at RT.

2， Resuspend latex beads in 3% BSA containing 25 mM Na3PO4 (pH 6.0) and incubate at RT for 15 min with bath sonication.
* Coating beads in BSA insures beads remain in a monodisperse state.

3， Wash the beads once with culture medium containing 5% FBS.

4 ，Resuspend the beads in culture medium at concentration 2.0%.This is beads stock. Stored in darkness at 4℃.

---Assay

1， Controls and samples: Intact control (No staining) 1 well
-Negative control (azide treated) 1 well
-Normal control 1 well
-sample 5 wells

* In order to differentiate between phagocytosed beads and beads nonspecifically adhere to the cell surface, control cells are exposed to 0,3%(w/v) azide for 10 min prior to the addition of coated beads. This treatment compromises microglial energetic processes and few beads were internalized as observed by fluorescent microscopy. Mean fluorescence of azide-treated microglia was used as the negative control and was subtracted from values obtained in experimental samples.

2， For experiments using 6 well-plates, 15 μl beads stock in 1 ml culture medium is applied to each well. Votex the beads stock well and take out 105 μl and add into 7 ml culture medium. Bath sonificate for 10 min at RT in darkness. This is beads working solution.

For experiments using 12 well-plates, 6 μl beads stock in 0.4 ml culture medium is applied to each well.

3， Wash each well twice with PBS and replace with beads working solution, 1 ml/well for 6-well plate, 0.4 ml/well for 12 well-plate. Incubate in the dark at 37 ℃ for 80-120 min.

4， Remove beads working solution and wash 3 times with PBS to remove excess beads.

5， Lift the cells by scrapping or trypsinization and wash the cells wish PBS.

6， Stain with PI (4ug/ml final concentration) and run for FACS.



此3D图是我的结果。

所用细胞并非专职吞噬细胞（astrocyte）。

随处理剂量增大，可见荧光细胞的比例以及荧光强度逐渐降低。

最下面灰色的细胞群是无荧光对照。


下图是Dot plot

图形特点：感叹号



下图是专职吞噬细胞（microglia）对荧光微球的吞噬。

可见所给处理对吞噬功能无明显影响。

Histogram

Dr.LinZhen

看不见图片诶~