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 Annexin V-FITC/PI Apoptosis Detection Kit 細(xì)胞凋亡檢測(cè)試劑盒

產(chǎn)品信息

產(chǎn)品描述

Annexin V-FITC/PI細(xì)胞凋亡檢測(cè)試劑盒是用FITC標(biāo)記的Annexin V作為探針,，來(lái)檢測(cè)細(xì)胞早期凋亡的發(fā)生,。

其檢測(cè)原理為：在正常的活細(xì)胞中,，磷脂酰絲氨酸（phosphotidylserine,，PS）位于細(xì)胞膜的內(nèi)側(cè)，但在早期凋亡的細(xì)胞中,，PS 從細(xì)胞膜的內(nèi)側(cè)翻轉(zhuǎn)到細(xì)胞膜的表面,，暴露在細(xì)胞外環(huán)境中,。Annexin-Ⅴ（膜聯(lián)蛋白-V）是一種分子量為35-36 kDa的Ca²⁺ 依賴性磷脂結(jié)合蛋白，能與PS高親和力結(jié)合,，可通過細(xì)胞外側(cè)暴露的磷脂酰絲氨酸與凋亡早期細(xì)胞的胞膜結(jié)合。

另外,，本試劑盒中還提供了碘化丙啶（Propidium Iodide,，PI）用來(lái)區(qū)分存活的早期細(xì)胞和壞死或晚期凋亡細(xì)胞。PI是一種核酸染料,，它不能透過正常細(xì)胞或早期凋亡細(xì)胞的完整的細(xì)胞膜,，但可以透過凋亡晚期和壞死細(xì)胞的細(xì)胞膜而使細(xì)胞核染紅。因此,，將Annexin V與PI聯(lián)合使用時(shí),，PI 則被排除在活細(xì)胞（Annexin V-/PI-）和早期凋亡細(xì)胞（Annexin V+/PI-）之外，而晚期凋亡細(xì)胞和壞死細(xì)胞同時(shí)被FITC 和PI 結(jié)合染色呈現(xiàn)雙陽(yáng)性（Annexin V+/PI+）,。

本試劑盒可用于流式細(xì)胞儀,、熒光顯微鏡進(jìn)行檢測(cè)。

產(chǎn)品組分

運(yùn)輸與保存方法

冰袋（wet ice）運(yùn)輸,。-20℃避光長(zhǎng)期保存,，避免反復(fù)凍融。

【注】：如果需要在短時(shí)間內(nèi)多次重復(fù)使用,，可以在4℃避光保存,，半年有效。

注意事項(xiàng)

1）由于細(xì)胞凋亡是一個(gè)快速的過程,，建議樣品在染色后1小時(shí)之內(nèi)進(jìn)行分析,。

2） 對(duì)于貼壁細(xì)胞，消化是一個(gè)關(guān)鍵步驟,。貼壁細(xì)胞誘導(dǎo)細(xì)胞凋亡時(shí)如有漂浮細(xì)胞,，需收集漂浮細(xì)胞和貼壁細(xì)胞后合并染色。處理貼壁細(xì)胞時(shí)要小心操作,，盡量避免人為的損傷,。胰酶消化時(shí)間過短，細(xì)胞需要用力吹打才能脫落,，容易造成細(xì)胞膜的損傷,；PI攝入過多，消化時(shí)間過長(zhǎng),，細(xì)胞膜同樣易造成損傷,，甚至?xí)绊懠?xì)胞膜上磷脂酰絲氨酸與Annexin V-FITC的結(jié)合。消化時(shí)將胰酶鋪滿孔板底后,，輕搖使胰酶與細(xì)胞充分接觸,，然后倒掉大部分胰酶,，利用剩余少量胰酶再消化一段時(shí)間，待細(xì)胞間空隙增大,，瓶底呈花斑狀即可終止,。在消化液中盡量不用EDTA，EDTA會(huì)影響Annexin V與PS的結(jié)合,。

3） 實(shí)驗(yàn)中如需要固定細(xì)胞,，比如在檢測(cè)凋亡的同時(shí)檢測(cè)細(xì)胞周期，只能選用Annexin V-FITC,，而不能選用Annexin V-EGFP,，因?yàn)樵诠潭ㄟ^程中EGFP會(huì)變性導(dǎo)致喪失激發(fā)熒光的能力。固定前需要先將細(xì)胞與Annexin V-FITC進(jìn)行孵育,，并用Binding buffer洗掉未結(jié)合的Annexin V-FITC,。因?yàn)楣潭ㄟ^程中細(xì)胞通透性增加會(huì)產(chǎn)生細(xì)胞碎片，可以和Annexin V結(jié)合,，對(duì)結(jié)果產(chǎn)生干擾,。

4）如果樣品來(lái)源于血液，請(qǐng)務(wù)必除去血液中的血小板,。因?yàn)檠“搴蠵S,，能與Annexin V結(jié)合，從而干擾實(shí)驗(yàn)結(jié)果?？梢允褂煤蠩DTA的緩沖劑并在200 g離心洗去血小板,。

5）試劑在開蓋前請(qǐng)短暫離心,，將蓋內(nèi)壁上的液體甩至管底,，避免開蓋時(shí)液體灑落。

6）Annexin V-FITC和PI是光敏物質(zhì),，在操作時(shí)請(qǐng)注意避光,。

操作方法

1.1 樣品染色

1）懸浮細(xì)胞：300 g,，4℃離心5 min收集細(xì)胞。

貼壁細(xì)胞：用不含EDTA的胰酶消化后,，300 g,，4℃離心5 min收集細(xì)胞。胰酶消化時(shí)間不宜過長(zhǎng),，以防引起假陽(yáng)性。

2）用預(yù)冷的PBS洗滌細(xì)胞2次,，每次均需300 g,，4℃離心5 min。收集1～5×10⁵細(xì)胞。

3）吸棄PBS,，加入100 μL 1×Binding Buffer重懸細(xì)胞。

4）加入5 μL Annexin V-FITC和10 μLPI Staining Solution,，輕輕混勻。

5）避光,、室溫反應(yīng)10-15 min,。

6）加入400 μL 1×Binding Buffer，混勻后放置于冰上,，樣品在1小時(shí)內(nèi)用流式細(xì)胞儀或熒光顯微鏡檢測(cè),。

【注】：為了避免洗滌細(xì)胞時(shí)損失細(xì)胞，在吸液時(shí)可以用大的Tip頭套上小的Tip頭吸液,。

1.2 樣品分析

A．流式細(xì)胞儀分析：

FITCzui大激發(fā)波長(zhǎng)為488 nm,，zui大發(fā)射波長(zhǎng)525 nm，F(xiàn)ITC的綠色熒光在FL1通道檢測(cè),；PI-DNA復(fù)合物的zui大激發(fā)波長(zhǎng)為535 nm,，zui大發(fā)射波長(zhǎng)為615 nm，PI的紅色熒光在FL2或FL3通道檢測(cè),。用CellQuest等軟件進(jìn)行分析,，繪制雙色散點(diǎn)圖（two-color dot plot），FITC為橫坐標(biāo),，PI為縱坐標(biāo),。典型的實(shí)驗(yàn)中，細(xì)胞可以分成三個(gè)亞群,，活細(xì)胞僅有很低強(qiáng)度的背景熒光,，早期凋亡細(xì)胞僅有較強(qiáng)的綠色熒光，晚期凋亡細(xì)胞有綠色和紅色熒光雙重染色,。

B．熒光顯微鏡分析：

1）滴一滴用Annexin V-FITC/PI雙染的細(xì)胞懸液于載玻片上,，并用蓋玻片蓋上細(xì)胞。

【注】：對(duì)于貼壁細(xì)胞,，可直接用蓋玻片培養(yǎng)細(xì)胞并誘導(dǎo)細(xì)胞凋亡,。

2）在熒光顯微鏡下用雙色濾光片觀察,。Annexin V-FITC熒光信號(hào)呈綠色,，PI熒光信號(hào)呈紅色。

數(shù)據(jù)展示

數(shù)據(jù)來(lái)源：上海交通大學(xué)納米生物工程研究所,；

文章信息：Cui, D., et al., Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying both Ligand and siRNA. Sci Rep, 2015. 5: p. 10726.

細(xì)胞類型：MGC803 cells,；

所用試劑：Yeasen，Cat：40302,，Annexin V-FITC/PI Apoptosis Detection Kit ,。

“Fig5.Determination of cell death by flow cytometry of Annexin V-FITC/PI staining in MGC803 cells transfected with 25 nM FA-pRNA-3WJ-BRCAA1siRNA or FA-pRNA-3WJ-Scram-siRNA for 48 h.”

數(shù)據(jù)來(lái)源：中山大學(xué)；

文章信息：Zhou, T., et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/beta-catenin signaling pathway in hippocampal neurons. Biochem Biophys Res Commun, 2017. 486(4): p. 1005-1013.

細(xì)胞類型：Hippocampal neurons,；

所用試劑：Yeasen,，Cat：40302 ，Annexin V-FITC/PI Apoptosis Detection Kit,。

“Fig. 3. Wnt/b-catenin mediates the expression levels of apoptosis-related proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis,。scatter diagram of PI/Annexin V gating from different groups. a. Control; b.OGD/R; c.OGD/R t MH(24 h); d.OGD/R t MH(24 h)tDkk1; e. OGD/R t Dkk1.”

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[4].Tong, K., C. Xin and W. Chen, Isoimperatorin induces apoptosis of the SGC-7901 human gastric cancer cell line via the mitochondria-mediated pathway. Oncol Lett, 2017. 13(1): p. 518-524.

[5].Tian, Y., et al., Tobacco Mosaic Virus-Based 1D Nanorod-Drug Carrier via the Integrin-Mediated Endocytosis Pathway. ACS Appl Mater Interfaces, 2016. 8(17): p. 10800-7.

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[8].Yu, H., et al., Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro. Exp Cell Res, 2016. 343(2): p. 218-22.

[9].Li, J., et al., Phenylethanoid Glycosides from Cistanche tubulosa Inhibits the Growth of B16-F10  Cells both in Vitro and in Vivo by Induction of Apoptosis via Mitochondria-dependent Pathway. J Cancer, 2016. 7(13): p. 1877-1887.

[10]. Yang, Y., et al., Human CIK Cells Loaded with Au Nanorods as a Theranostic Platform for Targeted Photoacoustic Imaging and Enhanced Immunotherapy and Photothermal Therapy. Nanoscale Res Lett, 2016. 11(1): p. 285.

[11]. Zhuang, Y.T., et al., IL-6 induced lncRNA MALAT1 enhances TNF-alpha expression in LPS-induced septic cardiomyocytes via activation of SAA3. Eur Rev Med Pharmacol Sci, 2017. 21(2): p. 302-309.

[12]. Wang, P., et al., Blood Plasma of Patients with Parkinsons Disease Increases Alpha-Synuclein Aggregation and Neurotoxicity. Parkinsons Dis, 2016. 2016: p. 7596482.

[13]. Ge, G.F., et al., Baicalein attenuates vinorelbine-induced vascular endothelial cell injury and chemotherapeutic phlebitis in rabbits. Toxicol Appl Pharmacol, 2017. 318: p. 23-32.

[14]. Ge, G.F., et al., Baicalein attenuates vinorelbine-induced vascular endothelial cell injury and chemotherapeutic phlebitis in rabbits. Toxicol Appl Pharmacol, 2017. 318: p. 23-32.

[15]. Cai, G., et al., Galectin-3 induces ovarian cancer cell survival and chemoresistance via TLR4 signaling activation. Tumour Biol, 2016. 37(9): p. 11883-11891.

[16]. Zhou, T., et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/beta-catenin signaling pathway in hippocampal neurons. Biochem Biophys Res Commun, 2017. 486(4): p. 1005-1013.

[17]. Cui, D., et al., Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying  both Ligand and siRNA. Sci Rep, 2015. 5: p. 10726.

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[19]. Wu, Z., et al., LncRNA-N1LR Enhances Neuroprotection Against Ischemic Stroke Probably by Inhibiting p53 Phosphorylation. Mol Neurobiol, 2016.

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