作者Cheinder (w23w)
看板Biotech
标题[求救] SH-SY5Y细胞用DCFH-DA 测ROS
时间Mon Mar 26 21:14:49 2012
各位前辈大家好!
我想请教一下在做ROS assay, 以流式细胞仪侦测或萤光显微镜发
现,加入 H202 的组别的强度几乎比不加药的控制组萤光低了十倍,
而加入样品与H202的组别萤光相差无几, H202处理的剂量是根据细
胞 70% 存活率 以下是我的实验条件
1. 2*10 5次方 SHSY-5Y Human Neuroblastoma Cells, 500 ul in
24 well 24 Hr
2. Reomove culture medium and preculture with sample for 1 Hr
3. Then, reomove medium and add fresh 100 uM H202 for 2 Hr
24 well 24 Hr
2. Reomove culture medium and preculture with sample for 1 Hr
3. Then, reomove medium and add fresh 100 uM H202 for 2 Hr
4. After incubation period, removal of medium and wash by PBS twice
5. Add 10 uM DCFHDA in serum-free medium for 30 min
6. After that, removal of DCFHDA and wash with PBS twice
7. Observe by fluorescence microscopy or trypsinized and centrifuge
for flow analysis
这个实验卡住很久, DCFHDA 也是新配 10 mM in DMSO stock, 在显微镜观察加入细
胞型态的完整性相对於控制组有明显差异, 神经突触消失, 但是以萤光显微镜看反倒
是控制组几乎每颗细胞都很亮, 加入 H202 组别 则萤光很弱,希望各位前辈给小弟
指点,感恩不尽!!!
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◆ From: 140.121.156.55
1F：推 logphase:H2O2放多久了?会不会过期了? 03/26 22:00
2F：→ Cheinder:H202是新买的 在4度C冰箱 每次使用都原液配 03/27 14:01
3F：推 ChesterYeah:说实在的~实验室目前也常遇到这麽麻烦~ 03/31 02:00