作者Cheinder (w23w)
看板Biotech
標題[求救] SH-SY5Y细胞用DCFH-DA 測ROS
時間Mon Mar 26 21:14:49 2012
各位前輩大家好!
我想請教一下在做ROS assay, 以流式細胞儀偵測或螢光顯微鏡發
現,加入 H202 的組別的強度幾乎比不加藥的控制組螢光低了十倍,
而加入樣品與H202的組別螢光相差無幾, H202處理的劑量是根據細
胞 70% 存活率 以下是我的實驗條件
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
這個實驗卡住很久, DCFHDA 也是新配 10 mM in DMSO stock, 在顯微鏡觀察加入細
胞型態的完整性相對於控制組有明顯差異, 神經突觸消失, 但是以螢光顯微鏡看反倒
是控制組幾乎每顆細胞都很亮, 加入 H202 組別 則螢光很弱,希望各位前輩給小弟
指點,感恩不盡!!!
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◆ From: 140.121.156.55
1F：推 logphase:H2O2放多久了?會不會過期了? 03/26 22:00
2F：→ Cheinder:H202是新買的 在4度C冰箱 每次使用都原液配 03/27 14:01
3F：推 ChesterYeah:說實在的~實驗室目前也常遇到這麼麻煩~ 03/31 02:00