Treg的破膜剂是什么成分

chujindong

CD4+CD25+FOXP3阳性的Treg的破膜剂究竟是什么成分？据说使用普通的破细胞膜的破膜膜剂不能破核膜，因此不能用来检测FOXP3。不知道除了买进口的试剂，大家有没有其他的方法来做这个？

niwanmao

chujindong 发表于 2011-10-27 16:38

                               
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CD4+CD25+FOXP3阳性的Treg的破膜剂究竟是什么成分？据说使用普通的破细胞膜的破膜膜剂不能破核膜，因此不能 ...

我们平时用的破膜剂是只能破到胞膜。FOXP3的破膜剂有两个组分：fix&perm和perm，前者应该是固定胞膜和破胞膜两个作用，后者应该是进入细胞内破核膜。至于配方，肯定是商业机密 。
应该有其它方法，你可以找一下foxp3的早期文章，里面肯定有，因为他们刚开始研究的时候不可能买商业化的试剂。

niwanmao

找到一篇早期文章了：http://onlinelibrary.wiley.com/doi/10.1002/eji.200526189/abstract
全文我看不到，但在一篇引用它的文章中看到了简要步骤，似乎利用0.5％吐温：
In brief, fresh PBMCs were washed in PBS, fixed in 1 ml of PBS with 1% paraformaldehyde and 0.05% Tween, and kept overnight at 4°C. Cells were treated twice with 0.5 ml of DNase at 100 Kunitz/ml according to manufacturer’s instructions (Sigma-Aldrich). Cells were incubated with mouse anti-human Foxp3 (clone150D/E4, a kind gift from Alison Banham, University of Oxford, Oxford, United Kingdom) for 1 hour at room temperature and washed with FACS buffer (PBS, 3% FCS, 0.5% Tween-20, and 0.05% azide). Foxp3 staining was detected by staining with Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes; Invitrogen). Cell-surface staining was performed using anti-CD4–PerCP (BD) and anti-CD25–PE (Dako) for 20 minutes at room temperature.

chujindong

niwanmao 发表于 2011-10-31 13:08

                               
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找到一篇早期文章了：http://onlinelibrary.wiley.com/doi/10.1002/eji.200526189/abstract
全文我看不到， ...

看着是用DNase处理的