沃吉基因---细胞发生铁死亡的检测方法大全

Ferroptotic特征

铁死亡的主要机制是，在铁的作用下，催化细胞膜上高浓度的不饱和脂肪酸，发生脂质体过氧化，从而诱导细胞死亡.

1)在细胞形态方面，铁死亡会导致细胞线粒体变小，膜密度增高，嵴减少。细胞核中形态变化不明显.

2)在细胞成分方面，铁死亡表现为脂质过氧化增高，ROS升高。也有一些特征基因发生变化.例如：谷胱甘肽过氧化物酶4(GPX4)

Jaeschke, Hartmut & Ramachandran, Anup & Chao, Xiaojuan & Ding, Wen-Xing. (2019). Emerging and established modes of cell death during acetaminophen-induced liver injury. Archives of Toxicology. 93. 1-12. 10.1007/s00204-019-02597-1.

细胞铁死亡主要的检测方法

细胞活性检测：

1).CCK8检测：可用于简便而准确的细胞增殖和毒性分析.

 (B) CCK-8 assay; the control group (I) was cultured in medium only, while the ferroptosis model group (II) was treated with erastin. The sample group was damaged by erastin and then treated with 0.3 (III) and 3.1 μM (IV) geraniin.

2).流式（分析细胞早期凋亡和晚期凋亡的比例） 

Ferroptosis-Inhibitory Effect and Possible Mechanisms of Ellagitannin Geraniin.B. Chen, X. Li, J. Liu, Y. Li, W. Dai, Y. Chen, D. Chen, ChemistryOpen 2021, 10, 737. 

铁含量的检测：

细胞内铁浓度的测定：通过添加酸性缓冲液而使样品释放铁。可直接测定样品中的Fe2+ 或将样品还原后测量总铁(Fe2+ 和Fe3+)。释放的铁与发色团反应，然后测量593 nm处的吸光度值，以计算铁离子水平。铁含量检测试剂盒提供了一种简单方便的铁含量测定方法.

The intracellular iron concentration was detected by iron ion kit

Song X, Wang X, Liu Z and Yu Z (2020) Role of GPX4-Mediated Ferroptosis in the Sensitivity of Triple Negative Breast Cancer Cells to Gefitinib. Front. Oncol. 10:597434. doi: 10.3389/fonc.2020.597434.

活性氧水平检测

1).用荧光显微镜观察细胞内的活性氧水平。

Song X, Wang X, Liu Z and Yu Z (2020) Role of GPX4-Mediated Ferroptosis in the Sensitivity of Triple Negative Breast Cancer Cells to Gefitinib. Front. Oncol. 10:597434. doi: 10.3389/fonc.2020.597434.

2).用流式细胞仪分析细胞活性氧水平。

Chen, Peng & Wu, Qibiao &. (2020). Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis. Signal Transduction and Targeted Therapy. 5. 51. 10.1038/s41392-020-0149-3. 

脂质过氧化物检测：GSH、MDA

MDA level and GSH level were measured using the Lipid Peroxidation Assay Kit (ab118970, Abcam) and the Glutathione Assay Kit (CS0260, Sigma, St. Louis, MO, USA), respectively.

Song X, Wang X, Liu Z and Yu Z (2020) Role of GPX4-Mediated Ferroptosis in the Sensitivity of Triple Negative Breast Cancer Cells to Gefitinib. Front. Oncol. 10:597434. doi: 10.3389/fonc.2020.597434.

电镜检测

通过电镜观察线粒体的变化：检测细胞形态，细胞发生铁死亡时线粒体变小，膜密度增高，嵴减少甚至消失.

Lewerenz, Jan & Ates, Gamze & Methner, Axel & Conrad, Marcus & Maher, Pamela. (2018). Oxytosis/Ferroptosis—(Re-) Emerging Roles for Oxidative Stress-Dependent Non-apoptotic Cell Death in Diseases of the Central Nervous System. Frontiers in Neuroscience. 12. 10.3389/fnins.2018.00214. 

细胞铁死亡相关基因表达水平变化

Ferroptosis PCR Array试剂盒检测样品中细胞铁死亡哪些基因发生变化.

Sun, S., Gao, T., Pang, B. et al. RNA binding protein NKAP protects glioblastoma cells from ferroptosis by promoting SLC7A11 mRNA splicing in an m6A-dependent manner. Cell Death Dis 13, 73 (2022). https://doi.org/10.1038/s41419-022-04524-2.