該步驟僅提供指南，應(yīng)根據(jù)您的特定需求進(jìn)行修改。在制備ROS Brite?工作溶液之前，根據(jù)需要處理細(xì)胞。
1）在DMSO中制備10至20mM ROS Brite TM APF（或HPF）儲(chǔ)備溶液。通過(guò)將DMSO儲(chǔ)備溶液稀釋到含有20mM Hepes緩沖液（HHBS）的Hanks溶液中，制備1至10μM的工作溶液。
2）根據(jù)需要處理細(xì)胞（例如，用50-100nM血管緊張素II處理RASM細(xì)胞3-5小時(shí)）
3）用ROS Brite?APF（或HPF）（1-10μM，步驟＃1）在37℃孵育細(xì)胞20-60分鐘。
4）用HHBS緩沖液替換染料加載溶液。
5）用適當(dāng)?shù)臒晒鈨x器在Ex / Em = 490 / 525mm（截止= 515nM）下用底部讀取模式分析細(xì)胞（例如，用于熒光顯微鏡的FITC濾光器，用于流式細(xì)胞儀的FL1濾光器）。
注意：BSA和酚紅會(huì)影響熒光，應(yīng)謹(jǐn)慎使用。 APF和HPF均可用于溶液測(cè)定或細(xì)胞內(nèi)測(cè)量。
參考文獻(xiàn)
1. Du M, Zhang D, Yan C, Zhang X. (2012) Developmental toxicity evaluation of three hexabromocyclododecane diastereoisomers on zebrafish embryos. Aquat Toxicol, 112-113, 1.
2. Park WH. (2012) MAPK inhibitors and siRNAs differentially affect cell death and ROS levels in arsenic trioxide-treated human pulmonary fibroblast cells. Oncol Rep, 27, 1611.
3. Park WH, Kim SH. (2012) MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion. Oncol Rep, 27, 1284.
4. Wu YT, Lin CY, Tsai MY, Chen YH, Lu YF, Huang CJ, Cheng CM, Hwang SP. (2011) beta-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos. J Biomed Sci, 18, 70.
5. You BR, Kim SZ, Kim SH, Park WH. (2011) Gallic acid-induced lung cancer cell death is accompanied by ROS increase and glutathione depletion. Mol Cell Biochem, 357, 295.