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标题：请问用全血和PBMC标Treg在标Foxp3时步骤有什么区别吗？ 回答者：niwanmao[
- 如果有表面标记，先进行表面标记。
- 全血，进行红细胞裂解，PBMC这一步可以省略。
- 进行FoxP3染色，详细步骤参见Kit的说明书，我这里附一份BioLegend的说明书：
- Prepare 1X working solutions of FOXP3 Fix/Perm buffer and FOXP3 Perm buffer prior to staining. The FOXP3 Fix/Perm buffer (4X) must be freshly diluted by diluting one (1) part FOPX3 Fix/Perm buffer (4X) with three (3) parts PBS. The FOXP3 Perm buffer (10X) should be diluted by diluting one (1) part FOXP3 Perm buffer (10X) with nine (9) parts of PBS.
- Add 1 ml of 1X FOXP3 Fix/Perm solution to each tube, vortex and incubate at room temperature in the dark for 20 minutes, then spin down the cells and remove supernatant.
- Wash once with cell staining buffer (Cat. No. 420201) by spin at 250 x g for 5 minutes and remove supernatant.
- Wash once with 1 ml 1X FOXP3 Perm buffer.
- Re-suspend cells in 1 ml 1X FOXP3 Perm buffer, incubate at room temperature in the dark for 15 minutes, spin down cells and discard the supernatant.
- Add 5 μl per tube of Alexa Fluor ® 488 anti-mouse/rat/human FOXP3 antibody, or Alexa Fluor ® 488 mouse IgG1, κ isotype control to the appropriate tubes, incubate at room temperature in the dark for 30 minutes.
- Wash twice with cell staining buffer, and re-suspend in 0.5 ml cell staining buffer then analyze with flow cytometer with appropriate instrument setting.
2009年8月12日 0:54 | 
发表于 2010-8-1 20:49:33
发表于 2020-5-7 16:01:41
