Annexin V-PE/7-AAD细胞凋亡检测试剂盒是用红色荧光染料PE（Phycoerythrin）标记的Annexin V作为探针，来检测细胞早期凋亡的发生，可用荧光显微镜、流式细胞仪或其他荧光检测设备进行检测。

其检测原理为：在正常的活细胞中，磷脂酰丝氨酸（phosphotidylserine，PS）位于细胞膜的内侧，但在早期凋亡的细胞中，PS 从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中。Annexin-Ⅴ（膜联蛋白-V）是一种分子量为35-36KD的Ca²⁺ 依赖性磷脂结合蛋白，能与PS高亲和力结合。可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。

另外，本试剂盒中提供的7-AAD可用来区分存活的早期细胞和坏死或晚期凋亡细胞。7-AAD是一种核酸染料，同PI有着相似的荧光特性，但其发射光谱较PI窄，对其他检测通道的干扰更小，在多色荧光分析中是PI的最佳替代品，可与Annexin V联合使用。该染料不能透过正常细胞或早期凋亡细胞的完整的细胞膜，但可穿透晚期凋亡细胞或者坏死细胞并与其内的DNA结合。因此将Annexin V-PE与7-AAD联合使用时，7-AAD则被排除在活细胞（Annexin V-/7-AAD-）和早期凋亡细胞（Annexin V+/7-AAD-）之外，而晚期凋亡细胞和坏死细胞同时被Annexin V-PE和7-AAD结合染色呈现双阳性（Annexin V+/7-AAD+）。

检测便捷、准确

冰袋（wet ice）运输。

本试剂盒中所有组分均在4 ℃保存，勿冰冻。Annexin V-PE、7-AAD需避光保存。一年有效。

Q:这两个试剂用什么通道检测？仪器是贝克曼的。

A: Annexin V-PE 的激发波长Ex=488nm；发射波长Em=578 nm，发出橙红色荧光，建议使用 FL2 通道检测；7-AAD 的激发波长 Ex=546 nm；发射波长 Em=647 nm，发出红色荧光， 建议使用 FL3 通道检测。

Q: Annexin V-PE 染色失败或者阳性率偏低。

A:首先要确定实验中使用的诱导剂是否能产生凋亡。可通过设定确切凋亡诱导效果的阳性药物对照来排除这一情况。Annexin V-PE 染色失败，最常见的实验操作原因是贴壁细胞消化不当。Annexin V 跟 PS 的结合需要 Ca2+，Binding Buffer 中含有 2.5 mM 的 Ca2+，含 EDTA 的胰酶消化会影响染色，建议使用无EDTA 的胰酶。若使用含EDTA 的胰酶，必须通过洗涤步骤彻底去除EDTA。用 PBS 洗涤细胞沉淀后，应尽量去除残余液体。残留PBS 中的磷酸根，会形成磷酸钙沉淀。Binding Buffer 的瓶盖要紧闭，防止空气中的 CO2 进入后形成碳酸钙沉淀，减少游离Ca2+，导致实验失败。接触其他缓冲液如吸取PBS 后不更换枪头也会导致游离的 Ca2+减少。一些细胞其细胞膜上 PS 的密度较低，染色效果很差，此时建议更换细胞株或者采用 TUNEL 试剂盒检测凋亡。如果是贴壁细胞，药物诱导后漂浮的细胞也要收集，这部分细胞往往是凋亡阳性的细胞，丢弃会造成阳性结果偏低。

Q: 假阳性

A: 实验中发现未经诱导凋亡的对照细胞经染色后Annexin V-PE/7-AAD 双阳性比例过高，造成这种结果的原因可能是细胞本身活力低。建议用台盼蓝染色计算细胞活力，阴性对照台盼蓝拒染的细胞应大于 95%。若细胞活力低，建议重新复苏细胞，通常刚复苏的细胞至少应传代 2- 3 次以后才能进行实验。另一可能是细胞操作不当引起，操作过程中吹打细胞过于剧烈，贴壁细胞消化过程中消化时间过长，均可能导致细胞膜破坏，出现假阳性。此外，一般情况下诱导几个小时就可以出现早期凋亡，因此通常不需要处理大于 48 h 以上；诱导时间过长会使营养物质耗尽，导致细胞状态差，假阳性结果偏高。

[1] Shi J, Ju R, Gao H, Huang Y, Guo L, Zhang D. Targeting glutamine utilization to block metabolic adaptation of tumor cells under the stress of carboxyamidotriazole-induced nutrients unavailability. Acta Pharm Sin B. 2022;12(2):759-773. doi:10.1016/j.apsb.2021.07.008(IF:11.614)

[2] Liang C, Zou T, Zhang M, et al. MicroRNA-146a switches microglial phenotypes to resist the pathological processes and cognitive degradation of Alzheimer's disease. Theranostics. 2021;11(9):4103-4121. Published 2021 Feb 19. doi:10.7150/thno.53418(IF:11.556)

[3] Li J, Li J, Yao Y, et al. Biodegradable electrospun nanofibrous platform integrating antiplatelet therapy-chemotherapy for preventing postoperative tumor recurrence and metastasis. Theranostics. 2022;12(7):3503-3517. Published 2022 Apr 24. doi:10.7150/thno.69795(IF:11.556)

[4] Yang Y, Qiao X, Huang R, et al. E-jet 3D printed drug delivery implants to inhibit growth and metastasis of orthotopic breast cancer. Biomaterials. 2020;230:119618. doi:10.1016/j.biomaterials.2019.119618(IF:10.273)

[5] Wang D, Zhang L, Hu A, et al. Loss of 4.1N in epithelial ovarian cancer results in EMT and matrix-detached cell death resistance. Protein Cell. 2021;12(2):107-127. doi:10.1007/s13238-020-00723-9(IF:10.164)

[6] Fan Z, Chen X, Liu L, et al. Association of the Polymorphism rs13259960 in SLEAR With Predisposition to Systemic Lupus Erythematosus. Arthritis Rheumatol. 2020;72(6):985-996. doi:10.1002/art.41200(IF:9.586)

[7] Xu Y, Hu Y, Xu T, et al. RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage. Cell Rep. 2021;37(3):109854. doi:10.1016/j.celrep.2021.109854(IF:9.423)

[8] Zhu C, Zhang L, Zhao S, et al. UPF1 promotes chemoresistance to oxaliplatin through regulation of TOP2A activity and maintenance of stemness in colorectal cancer. Cell Death Dis. 2021;12(6):519. Published 2021 May 21. doi:10.1038/s41419-021-03798-2(IF:8.469)

[9] Yang YL, Zhang Y, Li DD, et al. RNF144A functions as a tumor suppressor in breast cancer through ubiquitin ligase activity-dependent regulation of stability and oncogenic functions of HSPA2. Cell Death Differ. 2020;27(3):1105-1118. doi:10.1038/s41418-019-0400-z(IF:8.086)

[10] Yuan S, Zheng P, Sun X, et al. Hsa_Circ_0001860 Promotes Smad7 to Enhance MPA Resistance in Endometrial Cancer via miR-520h. Front Cell Dev Biol. 2021;9:738189. Published 2021 Nov 29. doi:10.3389/fcell.2021.738189(IF:6.684)

[11] Lian Y, Hao H, Xu J, Bo T, Wang W. Histone Chaperone Nrp1 Mutation Affects the Acetylation of H3K56 in Tetrahymena thermophila. Cells. 2022;11(3):408. Published 2022 Jan 25. doi:10.3390/cells11030408(IF:6.600)

[12] Cai B, Hou M, Zhang S, et al. Dual Targeting of Endoplasmic Reticulum by Redox-Deubiquitination Regulation for Cancer Therapy. Int J Nanomedicine. 2021;16:5193-5209. Published 2021 Jul 30. doi:10.2147/IJN.S321612(IF:6.400)

[13] Liu G, Liu B, Liu X, et al. ARID1B/SUB1-activated lncRNA HOXA-AS2 drives the malignant behaviour of hepatoblastoma through regulation of HOXA3. J Cell Mol Med. 2021;25(7):3524-3536. doi:10.1111/jcmm.16435(IF:5.310)

[14] He J, Zhao T, Liu L, et al. The -172 A-to-G variation in ADAM17 gene promoter region affects EGR1/ADAM17 pathway and confers susceptibility to septic mortality with sepsis-3.0 criteria. Int Immunopharmacol. 2022;102:108385. doi:10.1016/j.intimp.2021.108385(IF:4.932)

[15] Yin H, Wang H, Wang M, et al. CircTCF25 serves as a sponge for miR-206 to support proliferation, migration, and invasion of glioma via the Jak2/p-Stat3/CypB axis. Mol Carcinog. 2022;61(6):558-571. doi:10.1002/mc.23402(IF:4.784)

[16] Lin L, Li H, Shi D, et al. Depletion of C12orf48 inhibits gastric cancer growth and metastasis via up-regulating Poly r(C)-Binding Protein (PCBP) 1. BMC Cancer. 2022;22(1):123. Published 2022 Jan 31. doi:10.1186/s12885-022-09220-0(IF:4.430)

[17] Wang P, Zhang C, Li J, et al. Adipose-derived mesenchymal stromal cells improve hemodynamic function in pulmonary arterial hypertension: identification of microRNAs implicated in modulating endothelial function. Cytotherapy. 2019;21(4):416-427. doi:10.1016/j.jcyt.2019.02.011(IF:4.297)

[18] Cao K, Chen Y, Zhao S, et al. Sirt3 Promoted DNA Damage Repair and Radioresistance Through ATM-Chk2 in Non-small Cell Lung Cancer Cells. J Cancer. 2021;12(18):5464-5472. Published 2021 Jul 13. doi:10.7150/jca.53173(IF:4.207)

[19] Cao Y, Chu C, Li X, Gu S, Zou Q, Jin Y. RNA-binding protein QKI suppresses breast cancer via RASA1/MAPK signaling pathway. Ann Transl Med. 2021;9(2):104. doi:10.21037/atm-20-4859(IF:3.932)

[20] Jiang ZQ, Li MH, Qin YM, Jiang HY, Zhang X, Wu MH. Luteolin Inhibits Tumorigenesis and Induces Apoptosis of Non-Small Cell Lung Cancer Cells via Regulation of MicroRNA-34a-5p. Int J Mol Sci. 2018;19(2):447. Published 2018 Feb 2. doi:10.3390/ijms19020447(IF:3.687)

[21] Qin Y, Liang Y, Jiang G, Peng Y, Feng W. ACY-1215 suppresses the proliferation and induces apoptosis of chronic myeloid leukemia cells via the ROS/PTEN/Akt pathway [published online ahead of print, 2022 Jun 8]. Cell Stress Chaperones. 2022;10.1007/s12192-022-01280-2. doi:10.1007/s12192-022-01280-2(IF:3.667)

[22] Wu S, Luo C, Li F, Hameed NUF, Jin Q, Zhang J. Silencing expression of PHF14 in glioblastoma promotes apoptosis, mitigates proliferation and invasiveness via Wnt signal pathway. Cancer Cell Int. 2019;19:314. Published 2019 Nov 27. doi:10.1186/s12935-019-1040-6(IF:3.439)

[23] Dai H, Wang J, Huang Z, et al. LncRNA OIP5-AS1 Promotes the Autophagy-Related Imatinib Resistance in Chronic Myeloid Leukemia Cells by Regulating miR-30e-5p/ATG12 Axis. Technol Cancer Res Treat. 2021;20:15330338211052150. doi:10.1177/15330338211052150(IF:3.399)

[24] Wu S, Luo C, Hameed NUF, Wang Y, Zhuang D. UCP2 silencing in glioblastoma reduces cell proliferation and invasiveness by inhibiting p38 MAPK pathway. Exp Cell Res. 2020;394(1):112110. doi:10.1016/j.yexcr.2020.112110(IF:3.383)

[25] Chen A, Fang D, Ren Y, Wang Z. Matrine protects colon mucosal epithelial cells against inflammation and apoptosis via the Janus kinase 2 /signal transducer and activator of transcription 3 pathway. Bioengineered. 2022;13(3):6490-6499. doi:10.1080/21655979.2022.2031676(IF:3.269)

[26] Jiang K, Zou H. microRNA-20b-5p overexpression combing Pembrolizumab potentiates cancer cells to radiation therapy via repressing programmed death-ligand 1. Bioengineered. 2022;13(1):917-929. doi:10.1080/21655979.2021.2014617(IF:3.269)

[27] Liu S, Xin Y, Shi J, et al. ML365 inhibits lipopolysaccharide-induced inflammatory responses via the NF-κB signaling pathway. Immunobiology. 2022;227(3):152208. doi:10.1016/j.imbio.2022.152208(IF:3.144)

[28] Wang Z, Fu L, Zhang J, et al. A comprehensive analysis of potential gastric cancer prognostic biomarker ITGBL1 associated with immune infiltration and epithelial-mesenchymal transition. Biomed Eng Online. 2022;21(1):30. Published 2022 May 20. doi:10.1186/s12938-022-00998-5(IF:2.819)

[29] Sun Y, Tian X, Wu H, et al. H₂O₂ signaling modulates Glycoprotein-1 induced programmed cell death in tobacco suspension cells. Pestic Biochem Physiol. 2021;171:104697. doi:10.1016/j.pestbp.2020.104697(IF:2.751)

[30] Zhao L, Zhou R, Wang Q, Cheng Y, Gao M, Huang C. MicroRNA-320c inhibits articular chondrocytes proliferation and induces apoptosis by targeting mitogen-activated protein kinase 1 (MAPK1). Int J Rheum Dis. 2021;24(3):402-410. doi:10.1111/1756-185X.14053(IF:2.454)

[31] Yao T, Wang L, Ding ZF, Yin ZS. hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis. J Clin Lab Anal. 2022;36(4):e24134. doi:10.1002/jcla.24134(IF:2.352)

[32] Zhong W, Zhao Y, Tian Y, Chen M, Lai X. The protective effects of HGF against apoptosis in vascular endothelial cells caused by peripheral vascular injury. Acta Biochim Biophys Sin (Shanghai). 2018;50(7):701-708. doi:10.1093/abbs/gmy048(IF:2.224)

[33] Zhang Z, Yi J, Xie B, et al. Parkin, as a Regulator, Participates in Arsenic Trioxide-Triggered Mitophagy in HeLa Cells. Curr Issues Mol Biol. 2022;44(6):2759-2771. Published 2022 Jun 20. doi:10.3390/cimb44060189(IF:2.081)

[34] Fan Y, Tan D, Zhang X, et al. Nuclear Factor-κB Pathway Mediates the Molecular Pathogenesis of LMNA-Related Muscular Dystrophies. Biochem Genet. 2020;58(6):966-980. doi:10.1007/s10528-020-09989-4(IF:2.027)

[35] Li M, Min W, Wang J, et al. Effects of mevalonate kinase interference on cell differentiation, apoptosis, prenylation and geranylgeranylation of human keratinocytes are attenuated by farnesyl pyrophosphate or geranylgeranyl pyrophosphate. Exp Ther Med. 2020;19(4):2861-2870. doi:10.3892/etm.2020.8569(IF:1.785)

[36] Zhao L. Protective effects of trimetazidine and coenzyme Q10 on cisplatin-induced cardiotoxicity by alleviating oxidative stress and mitochondrial dysfunction. Anatol J Cardiol. 2019;22(5):232-239. doi:10.14744/AnatolJCardiol.2019.83710(IF:1.112)