[求救] SH-SY5Y细胞用DCFH-DA 測ROS

看板Biotech (生命科學)作者Cheinder (w23w)時間14年前 (2012/03/26 21:14)推噓2(2推 0噓 1→)

留言3則, 3人參與討論串1/1

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!!! -- 昂首千丘遠嘯傲風間堪尋敵手共論劍高處不勝寒 -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 140.121.156.55