FOXP3以及IL-17——大鼠的外周血流式

倪shunlan

老师好！请问：1、大鼠外周血流式检测Foxp3以及IL-17时，我的固定/破膜剂使用的是ebioscience的Foxp3 Fixation/Permeabilization kit，请问大鼠检测细胞核内Foxp3因子时需破膜孵育几小时左右效果更好？（之前看过网站上有人分享：人的孵育30~60min，小鼠可以延长到18h，不知大鼠几小时更合适？）
2、检测细胞内因子IL-17时，ebioscience的固定/破膜剂孵育多久合适？
希望各位做过大鼠此方面流式的老师，不吝赐教！谢谢！

田田

顶一下，我也想做这个，还有CD4和CD8.

niwanmao

找找文献看：


1篇：http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3275433/
For intracellular cytokine staining, cells were pretreated for 4 h with 50 ng/ml PMA, 1 µg/ml ionomycin, and 1 µg/ml brefeldin A (Sigma-Aldrich), then washed, fixed, permeabilized overnight with Cytofix/Cytoperm buffer (eBioscience), and intracellularly stained with Abs against IFN-γ and IL-17.


2篇：http://www.plosone.org/article/i ... ournal.pone.0049008
For intracellular staining cells were fixed and permeabilized according to the manufacturer's instructions for the rat anti-mouse/rat Foxp3 staining kit (eBioscience, Germany) and subsequently stained with mouse anti-rat IFN-γ antibodies (clone DB-1) conjugated with FITC, PE or Alexa Fluor 647, rat anti-mouse IL-17 antibodies crossreactive with rat IL-17 (clone TC11-18H10.1) conjugated with FITC or PerCP/Cy5.5 (both BioLegend/Biozol, Germany) or mouse anti-rat IL-10 (clone A5-4, BD Biosciences, Germany) conjugated with PE or the respective mouse IgG2b isotype control for anti-IL-10 (clone MCP-11, BioLegend/Biozol, Germany). Mouse anti-rat CD68 conjugated with Alexa Fluor 647 (clone ED1, AbD Serotec, Germany) was used for both, surface and intracellular staining.