适用仪器

样品实验方案

简要概述

1.准备密度为0.5-1×10⁶细胞/ mL的细胞
2.在0.5 mL细胞悬液中加入1 µL 500X Amplite ROS Green
3.在37℃下将细胞染色1小时
4.处理细胞以诱导ROS
5.使用带有FL1通道的流式细胞仪分析细胞（Ex / Em = 490/520 nm）

溶液配制

1.储备溶液配制

        除非另有说明，否则所有未使用的储备溶液应分成一次性等分试样，并在制备后储存在-20°C。 避免反复冻融循环。

Amplite ROS绿色储备溶液（500X）：将100 µL DMSO（组分C）添加到Amplite ROS Green（组分A）的小瓶中，并充分混合以制成500X Amplite ROS Green储备液。 避光。 注意：要存放，请密封管。有关细胞样品制备的指南，请点击查看。

操作步骤

1.对于每个样品，以0.5×10⁵至1×10⁶细胞/ mL的密度在0.5 mL测定缓冲液（组分B）或自备缓冲液中制备细胞。注意：应单独评估每个细胞系，以确定诱导ROS的最佳细胞密度。

2.将1 µL 500X Amplite ROS Green储备溶液加入0.5 mL细胞悬液中。

3.在37ºC下孵育1小时。注意：对于贴壁细胞，用0.5 mM EDTA轻轻提起细胞以保持细胞完整，并在与Amplite ROS Green孵育之前用含血清的培养基洗涤细胞一次。合适的孵育时间取决于所用的单个细胞类型和测试化合物，优化每个实验的孵育时间。

4.通过在所需的缓冲液（例如PBS或HBSS）中添加50 µL 11X测试化合物来处理细胞。对于对照孔（未处的细胞），添加相应量的缓冲液。

5.将细胞在37ºC下孵育，以诱导ROS（避光）。注意：我们在37℃下用100 µM TBHP（氢过氧化叔丁基）处理Jurkat细胞30分钟，以诱导ROS。有关详细信息，请参见图1。

6.使用具有FL1通道（Ex / Em = 490/520 nm）的流式细胞仪检测荧光强度。

图示

参考文献

Anti-proliferation effect of blue light-emitting diodes against antibiotic-resistant Helicobacter pylori
Authors: Ma, Jianwei and Hiratsuka, Takahiro and Etoh, Tsuyoshi and Akada, Junko and Fujishima, Hajime and Shiraishi, Norio and Yamaoka, Yoshio and Inomata, Masafumi
Journal: Journal of Gastroenterology and Hepatology (2017)

Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug Design, Development and Therapy (2017): 3343

Good hydration and cell-biological performances of superparamagnetic calcium phosphate cement with concentration-dependent osteogenesis and angiogenesis induced by ferric iron
Authors: Zhang, J and Shi, HS and Liu, JQ and Yu, T and Shen, ZH and Ye, JD
Journal: Journal of Materials Chemistry B (2015): 8782–8795

Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes
Authors: Patrick, Ping and Price, Tulin O and Diogo, Ana L and Sheibani, Nader and Banks, William A and Shah, Gul N
Journal: Journal of endocrinology and diabetes (2015)

Down-regulated peroxisome proliferator-activated receptor γ (PPARγ) in lung epithelial cells promotes a PPARγ agonist-reversible proinflammatory phenotype in chronic obstructive pulmonary disease (COPD)
Authors: Lakshmi, Sowmya P and Reddy, Aravind T and Zhang, Yingze and Sciurba, Frank C and Mallampalli, Rama K and Duncan, Steven R and Reddy, Raju C
Journal: Journal of Biological Chemistry (2014): 6383–6393

Superoxide dismutase as a target of clioquinol-induced neurotoxicity
Authors: Kawamura, Kazuyuki and Kuroda, Yukiko and Sogo, Masako and Fujimoto, Miki and Inui, Toshio and Mitsui, Takao
Journal: Biochemical and biophysical research communications (2014): 181–185

Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice
Authors: Nomura, Johji and Busso, Nathalie and Ives, Annette and Matsui, Chieko and Tsujimoto, Syunsuke and Shirakura, Takashi and Tamura, Mizuho and Kobayashi, Tsunefumi and So, Alex and er and Yamanaka, Yoshihiro
Journal: Scientific reports (2014): 4554

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: implications for cerebral microvascular disease in diabetes
Authors: Shah, Gul N and Morofuji, Yoichi and Banks, William A and Price, Tulin O
Journal: Biochemical and biophysical research communications (2013): 354–358

Automatic flow injection based methodologies for determination of scavenging capacity against biologically relevant reactive species of oxygen and nitrogen
Authors: Magalhaes LM, Lucio M, Segundo MA, Reis S, Lima JL.
Journal: Talanta (2009): 1219

Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species
Authors: Amaral S, Oliveira PJ, Ramalho-Santos J.
Journal: Curr Diabetes Rev (2008): 46

相关产品