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

產(chǎn)品信息

產(chǎn)品描述

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

其檢測原理為：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，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)胞同時被FITC 和PI 結(jié)合染色呈現(xiàn)雙陽性（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。

產(chǎn)品組分

運輸與保存方法

冰袋（wet ice）運輸。-20℃避光長期保存，避免反復(fù)凍融。

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

注意事項

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

2） 對于貼壁細(xì)胞，消化是一個關(guān)鍵步驟。貼壁細(xì)胞誘導(dǎo)細(xì)胞凋亡時如有漂浮細(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)胞時要小心操作，盡量避免人為的損傷。胰酶消化時間過短，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，消化時間過長，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會影響Annexin V與PS的結(jié)合。

3） 實驗中如需要固定細(xì)胞，比如在檢測凋亡的同時檢測細(xì)胞周期，只能選用Annexin V-FITC，而不能選用Annexin V-EGFP，因為在固定過程中EGFP會變性導(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。因為固定過程中細(xì)胞通透性增加會產(chǎn)生細(xì)胞碎片，可以和Annexin V結(jié)合，對結(jié)果產(chǎn)生干擾。

4）如果樣品來源于血液，請務(wù)必除去血液中的血小板。因為血小板含有PS，能與Annexin V結(jié)合，從而干擾實驗結(jié)果?？梢允褂煤蠩DTA的緩沖劑并在200 g離心洗去血小板。

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

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

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。胰酶消化時間不宜過長，以防引起假陽性。

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小時內(nèi)用流式細(xì)胞儀或熒光顯微鏡檢測。

【注】：為了避免洗滌細(xì)胞時損失細(xì)胞，在吸液時可以用大的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)波長為488 nm，zui大發(fā)射波長525 nm，F(xiàn)ITC的綠色熒光在FL1通道檢測；PI-DNA復(fù)合物的zui大激發(fā)波長為535 nm，zui大發(fā)射波長為615 nm，PI的紅色熒光在FL2或FL3通道檢測。用CellQuest等軟件進(jìn)行分析，繪制雙色散點圖（two-color dot plot），FITC為橫坐標(biāo)，PI為縱坐標(biāo)。典型的實驗中，細(xì)胞可以分成三個亞群，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熒光信號呈綠色，PI熒光信號呈紅色。

數(shù)據(jù)展示

數(shù)據(jù)來源：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.”

參考文獻(xiàn)

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[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.

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