Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒的操作方法有哪些？

发表于2025年2月8日由上海金畔生物科技有限公司

Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒是用于检测悬浮细胞和贴壁细胞的凋亡试剂盒。那么你知道Annexin V-FITC / PI细胞凋亡检测试剂盒的操作方法有哪些吗？让我们一起来看看吧！

一步法

1．细胞按照实验方案进行凋亡诱导，300 ×g 离心5 min，弃上清，收集细胞，PBS洗涤一次，轻轻重悬细胞并计数。

2．取 1~5 × 10⁵重悬的细胞，300 ×g 离心 5 min，弃上清。用 PBS 洗涤细胞一次，离心后弃上清，加入500 μL稀释的1 × Annexin V Binding Buffer重悬细胞。

3．细胞悬液中加入5μL的Annexin V-FITC Reagent和5μL的 PI Reagent (50μg/mL)。

4．轻柔涡旋混匀后，室温避光孵育15~20 min。

5．立即上机检测。如不能及时检测，请于冰上避光静置并于1小时内完成检测。

注：流式细胞仪检测时Annexin V-FITC可用FITC通道，PI 优先选择PerCP/Cy5.5 通道，其次是ECD通道。

两步法

1. 细胞按照实验方案进行凋亡诱导，300 ×g离心5 min，弃上清，收集细胞，PBS 洗涤一次，轻轻重悬细胞并计数。

2. 取1~5 × 10⁵重悬的细胞，300 ×g离心 5 min，弃上清。用 PBS 洗涤细胞一次，离心后弃上清，加入100 μL 稀释的 1 × Annexin V Binding Buffer 重悬细胞。

3. 细胞悬液中加入 2.5 μL 的 Annexin V-FITC Reagent 和2.5 μL的PI Reagent (50μg/mL)。（由于两步法分辨率更高，染色液用量减半依然可得到媲美一步法的效果；用户亦可根据自己的模型进行滴定后加入适量的染色液，用更少的量获得高质量的结果。）

4. 轻柔涡旋混匀后，室温避光孵育 15~20 min。

5. 加入 400 μL 稀释的1 × Annexin V Binding Buffer，混匀样本。

6. 立即上机检测。如不能及时检测，请于冰上避光静置并于1小时内完成检测。注：流式细胞仪检测时Annexin V-FITC 可用 FITC 通道，PI优先选择 PerCP/Cy5.5 通道，其次是 ECD 通道。

更多有关Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒的操作方法，请联系Elabscience试剂盒代理——上海金畔生物科技有限公司。

Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒的检测原理与注意事项

发表于2025年2月8日由上海金畔生物科技有限公司

Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒的检测原理与注意事项

Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒是用于检测悬浮细胞和贴壁细胞的凋亡试剂盒。那么你知道Annexin V-FITC / PI细胞凋亡检测试剂盒的检测原理与注意事项吗？让我们一起来看看吧！

一、检测原理

Annexin V 是一种钙离子依赖性磷脂结合蛋白，与磷脂酰丝氨酸 (PS) 有高度亲和力。当细胞发生凋亡时，膜内侧的磷脂酰丝氨酸 (PS) 外翻到膜表面，而被荧光染料 FITC 标记的 Annexin V 结合，可通过流式细胞仪或荧光显微镜进行检测。

由于凋亡晚期或坏死细胞膜丧失完整性，而碘化丙啶（Propidium Iodide, PI）可与双链 DNA 特异性结合并产生强烈的荧光，与 Annexin V 搭配使用，可区分处于不同凋亡时期的细胞。本试剂盒检测喜树碱诱导的 Jurkat 细胞凋亡效果如下图所示：

Jurkat细胞用5 μM 喜树碱（Camptothecin）(左) 或未加药 (右) 处理 4 h，本试剂盒染色后流式检测。Annexin V-FITC单阳细胞为早期凋亡细胞，Annexin V-FITC和PI双阳细胞为坏死或晚期凋亡细胞，PI 单阳细胞为裸核细胞。

二、注意事项

1. 检测贴壁细胞时，需收集诱导凋亡后产生的悬浮细胞，并与后续收集的贴壁细胞一起检测。

2. 应尽量避免消化贴壁细胞带来的机械损伤。同时，胰酶的消化液中应尽量不含EDTA，因为EDTA会影响Annexin V与磷脂酰丝氨酸的结合。

3. 如果使用含EDTA 的胰酶，收集细胞后应充分清洗，确保 EDTA 被去除干净。

4. 染色后宜尽快检测，时间过长可能会导致凋亡或坏死细胞的数量增加。

5. 荧光物质均易发生淬灭，在进行荧光观察时，尽量缩短观察时间，同时在操作和存放过程中也尽量注意避光保存。

更多有关Elabscience Annexin V-FITC / PI细胞凋亡检测试剂盒的检测原理与注意事项，请联系Elabscience试剂盒代理——上海金畔生物科技有限公司。

重点推介：Elabscience Annexin V荧光双染凋亡试剂

发表于2025年2月6日由上海金畔生物科技有限公司

重点推介：Elabscience Annexin V荧光双染凋亡试剂

产品名称：Elabscience Annexin V荧光双染凋亡试剂
产品简介：Annexin V探针是一种zui常用来检测细胞凋亡的方法。Annexin V是一种分子量为35~36kD的Ca²⁺依赖性磷脂结合蛋白，能与磷脂酰丝氨酸（PS）高亲和力特异性结合。在细胞凋亡早期，只分布在正常细胞膜脂质双层内侧的磷脂酰丝氨酸（PS）翻向外侧，用标记了荧光素的Annexin V作为荧光探针，并搭配Propidium iodide(PI)、7-AAD、DAPI等核酸染料使用，能够准确区分正常细胞、凋亡早期和凋亡晚期细胞。

Elabscience自主研发的Annexin V荧光双染细胞凋亡检测试剂盒，15种荧光与3种核酸染料组合，满足您的多种需求。

基本信息

应用类型	细胞凋亡-Annexin V
检测方法	荧光
样本类型	细胞
检测时间	40min
检测仪器	流式细胞仪
自备试剂	PBS(E-BC-R187), 去离子水
保存温度	2~8°C, shading light
运输条件	冰袋

产品优势

高准确性：无需固定，避免假阳性，阴阳性分群明显
多种选择： 15种荧光与3种核染料搭配
操作简便：一步法或两步法操作，15-20min即可完成染色
性价比高：自主研发，品质溯源，价格更实惠

Elabscience Annexin V荧光双染凋亡试剂产品列表如下：

荧光	货号	产品名称
FITC	E-CK-A211	Annexin V-FITC / PI荧光双染细胞凋亡检测试剂盒
FITC	E-CK-A211B	Annexin V-FITC / PI荧光双染细胞凋亡检测试剂盒
FITC	E-CK-A212	Annexin V-FITC / 7-AAD荧光双染细胞凋亡检测试剂盒
FITC	E-CK-A252	Annexin V-FITC / DAPI荧光双染细胞凋亡检测试剂盒
PE	E-CK-A216	Annexin V-PE / 7-AAD荧光双染细胞凋亡检测试剂盒
APC	E-CK-A217	Annexin V-APC / PI荧光双染细胞凋亡检测试剂盒
APC	E-CK-A218	Annexin V-APC / 7-AAD荧光双染细胞凋亡检测试剂盒
APC	E-CK-A258	Annexin V-APC / DAPI荧光双染细胞凋亡检测试剂盒

上海金畔生物科技有限公司代理Elabscience Annexin V荧光双染凋亡试剂，欢迎选购！

细胞凋亡试剂盒（PE和7-ADD标记）PE Annexin V Apoptosis DetectioBD 559763

发表于2025年1月24日由上海金畔生物科技有限公司

细胞凋亡试剂盒（PE和7-ADD标记）PE Annexin V Apoptosis Detectio

简要描述：

NameAnnexin V : PE Apoptosis Detection Kit IContentsAnnexin V-PE, 7-AAD, and Annexin V Binding BufferSize100 TestsRegulatory Status RUO

*，现货*格优惠，咨询

Technical Data Sheet

PE Annexin V Apoptosis Detection Kit I

Product Information

Material Number: 559763

Component: 51-66121E

Description: 10X Annexin V Binding Buffer

Size: 50 ml （1 ea）

Storage Buffer: Aqueous buffered solution containing no preservative.

Component: 51-68981E

Description: 7-AAD

Size: 2.0 ml （1 ea）

Vol. per Test: 5 μl

Storage Buffer: Aqueous buffered solution containing fetal bovine serum and ≤0.09% sodium

azide.

Component: 51-65875X

Description: PE Annexin V

Size: 0.5 ml （1 ea）

Vol. per Test: 5 μl

Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.

Description

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The

apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment,

condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features.

In apoptotic cells, the membrane phospholipid phosphatidylserine （PS） is translocated from the inner to the outer leaflet of the plasma

membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding

protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including

Phycoerythrin （PE）. This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are

undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, PE Annexin V staining can identify apoptosis at an

earlier stage than assays based on nuclear changes such as DNA fragmentation.

PE Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either

apoptotic or necrotic processes. Therefore, staining with PE Annexin V is typically used in conjunction with a vital dye such as

7-Amino-Actinomycin （7-AAD） to allow the investigator to identify early apoptotic cells （7-AAD negative, PE Annexin V positive）. Viable

cells with intact membranes exclude 7-AAD, wheras the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells

that are considered viable are PE Annexin V and 7-AAD negative; cells that are in early apoptosis are PE Annexin V positive and 7-AAD

negative; and cells that are in late apoptosis or already dead are are both PE Annexin V and 7-AAD positive. This assay does not distinguish

between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead

cells will stain with both PE Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from PE

Annexin V and 7-AAD negative （viable, or no measurable apoptosis）, to PE Annexin V positive and 7-AAD negative （early apoptosis,

membrane integrity is present） and finally to PE Annexin V and 7-AAD positive （end stage apoptosis and death）. The movement of cells

through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both PE Annexin V and 7-AAD

positive, in of itself, reveals less information about the process by which the cells underwent their demise.

559763 Rev. 8 Page 1 of 3

Flow Cytometric Analysis of PE Annexin V staining. Jurkat cells

（Human T-cell leukemia; ATCC TIB-152） were left untreated （top

panels） or treated for 4 hours with 4 μM Camptothecin （bottom

panels）. Cells were incubated with PE Annexin V in a buffer

containing 7-Amino-Actinomycin （7-AAD） and analyzed by flow

cytometry. Untreated cells were primarily PE Annexin V and 7-AAD

negative, indicating that they were viable and not undergoing

apoptosis. After a 4 hour treatment （bottom panels）, there were

primarily two populations of cells: Cells that were viable and not

undergoing apoptosis （PE Annexin V and 7-AAD negative）; cells

undergoing apoptosis （PE Annexin V positive and 7-AAD negative）.

A minor population of cells were observed to be PE Annexin V and

7-AAD positive, indicating that they were in end stage apoptosis or

already dead.

Preparation and Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Application Notes

Application

Flow cytometry Routinely Tested

Recommended Assay Procedure:

PE Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces （Kd of ~5 x 10^-2） with a

higher affinity for phosphatidylserine （PS） than most other phospholipids. PE Annexin V binding is calcium dependent and defined calcium and

salt concentrations are required for optimal staining as described in the PE Annexin V Staining Protocol. Investigators should note that PE

Annexin V flow cytometric analysis on adherent cell types （e.g HeLa, NIH 3T3, etc.） is not routinely tested as specific membrane damage

may occur during cell detachment or harvesting. Methods for utilizing Annexin V for flow cytometry on adherent cell types, however,

have been previously reported （Casiola-Rosen et al. and van Engelend et al.）.

INDUCTION OF APOPTOSIS BY CAMPTOTHECIN

The following protocol is provided as an illustration on how PE Annexin V may be used on a cell line （Jurkat）.

Materials

1. Prepare Camptothecin stock solution （Sigma-Aldrich Cat. No. C-9911）: 1 mM in DMSO.

2. Jurkat T cells （ATCC TIB-152）.

Procedure

1. Add Camptothecin （final conc. 4-6 μM） to 1 x 10^6 Jurkat cells.

2. Incubate the cells for 4-6 hr at 37°C.

3. Proceed with the PE Annexin V Staining Protocol to measure apoptosis.

PE ANNEXIN V STAINING PROTOCOL

PE Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on

the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid

phosphatidylserine （PS） is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external

environment. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying

apoptotic cells with exposed PS. 7-Amino-Actinomycin （7-AAD） is a standard flow cytometric viability probe and is used to distinguish viable

from nonviable cells. Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to

7-AAD. Cells that stain positive for PE Annexin V and negative for 7-AAD are undergoing apoptosis. Cells that stain positive for both PE

Annexin V and 7-AAD are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both PE

Annexin V and 7-AAD are alive and not undergoing measurable apoptosis.

559763 Rev. 8 Page 2 of 3

Reagents

1. PE Annexin V （component no. 51-65875X）: Use 5 μl per test.

2. 7-Amino-Actinomycin （7-AAD）（component no. 51-68981E） is a convenient, ready-to-use nucleic acid dye. Use 5 μl per test.

3. 10X Annexin V Binding Buffer （component no. 51-66121E）: 0.1 M Hepes/NaOH （pH 7.4）, 1.4 M NaCl, 25 mM CaCl2. For a 1X working

solution, dilute 1 part of the 10X Annexin V Binding Buffer to 9 parts of distilled water.

Staining

1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10^6 cells/ml.

2. Transfer 100 μl of the solution （1 x 10^5 cells） to a 5 ml culture tube.

3. Add 5 μl of PE Annexin V and 5 μl 7-AAD.

4. Gently vortex the cells and incubate for 15 min at RT （25°C） in the dark.

5. Add 400 μl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.

SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY

The following controls are used to set up compensation and quadrants:

1. Unstained cells.

2. Cells stained with PE Annexin V （no 7-AAD）.

3. Cells stained with 7-AAD （no PE Annexin V）.

Other Staining Controls:

A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with PE Annexin V and/or PE

Annexin V and 7-AAD. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in

the absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis （PE Annexin V

positive, 7-AAD negative or PE Annexin V positive, 7-AAD positive）.

The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo

apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from percentage of apoptotic cells in the

treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged

membrane and stain positive for 7-AAD as well as for PE Annexin V. Thus the assay does not distinguish between cells that have already

undergone an apoptotic cell death and those that have died as a result of necrotic pathway, because in either case the dead cells will stain with

both PE Annexin V and 7-AAD.

Product Notices

This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-μl experimental

sample （a test）.

2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.

Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before

discarding to avoid accumulation of potentially explosive deposits in plumbing.

4. Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.

References

Andree HA, Reuingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM. Binding of vascular anticoagulant alpha （VAC alpha） to planar

phospholipid bilayers. J Biol Chem. 1990; 265（9）:4923-4928. （Biology）

Casciola-Rosen L, Rosen A, Petri M, Schlissel M. Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events

and antigenic spread in systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1996; 93（4）:1624-1629. （Biology）

Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reuingsperger CP, Roos D. Human neutrophils lose their surface Fc gamma RIII and acquire

Annexin V binding sites during apoptosis in vitro. Blood. 1995; 85（2）:532-540. （Biology）

Koopman G, Reuingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on

B cells undergoing apoptosis. Blood. 1994; 84（5）:1415-1420. （Biology）

Martin SJ, Reuingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of

the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995; 182（5）:1545-1556. （Biology）

Raynal P, Pollard HB. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins.

Biochim Biophys Acta. 1994; 1197（1）:63-93. （Biology）

van Engeland M, Ramaekers FC, Schutte B, Reuingsperger CP. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent

cells in culture. Cytometry. 1996; 24（2）:131-139. （Biology）

Vermes I, Haanen C, Steffens-Nakken H, Reuingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early

apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995; 184（1）:39-51. （Biology）

559763

BD细胞凋亡试剂盒FITC Annexin V Apoptosis Detection Kit IBD556547

发表于2025年1月24日由上海金畔生物科技有限公司

BD细胞凋亡试剂盒FITC Annexin V Apoptosis Detection Kit I

简要描述：

ContentsAnnexin V-FITC, Propidium Iodide Staining Solution, Annexin V Binding BufferSize100 TestsRegulatory Status RUO
*，*，*，咨询

Description

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The

apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment,

condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features.

In apoptotic cells, the membrane phospholipid phosphatidylserine （PS） is translocated from the inner to the outer leaflet of the plasma

membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding

protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including FITC.

This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing

apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, FITC Annexin V staining can identify apoptosis at an earlier

stage than assays based on nuclear changes such as DNA fragmentation.

FITC Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either

apoptotic or necrotic processes. Therefore, staining with FITC Annexin V is typically used in conjunction with a vital dye such as propidium

iodide （PI） or 7-Amino-Actinomycin （7-AAD） to allow the investigator to identify early apoptotic cells （PI negative, FITC Annexin V

positive）. Viable cells with intact membranes exclude PI, wheras the membranes of dead and damaged cells are permeable to PI. For example,

cells that are considered viable are FITC Annexin V and PI negative; cells that are in early apoptosis are FITC Annexin V positive and PI

negative; and cells that are in late apoptosis or already dead are are both FITC Annexin V and PI positive. This assay does not distinguish

between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead

cells will stain with both FITC Annexin V and PI. However, when apoptosis is measured over time, cells can be often tracked from FITC

Annexin V and PI negative （viable, or no measurable apoptosis）, to FITC Annexin V positive and PI negative （early apoptosis, membrane

integrity is present） and finally to FITC Annexin V and PI positive （end stage apoptosis and death）. The movement of cells through these three

stages suggests apoptosis. In contrast, a single observation indicating that cells are both FITC Annexin V and PI positive, in of itself, reveals

less information about the process by which the cells underwent their demise.

Preparation and Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

556547 Rev. 5 Page 1 of 3

Flow Cytometric Analysis of FITC Annexin V staining. Jurkat cells

（Human T-cell leukemia; ATCC TIB-152） were left untreated （top

panels） or treated for 4 hours with 12 μM campotothecin （bottom

panels）. Cells were incubated with FITC Annexin V in a buffer

containing propidium iodide （PI） and analyzed by flow cytometry.

Untreated cells were primarily FITC Annexin V and PI negative,

indicating that they were viable and not undergoing apoptosis. After a

4 hour treatment （bottom panels）, there were primarily two

populations of cells: Cells that were viable and not undergoing

apoptosis （FITC Annexin V and PI negative） and cells undergoing

apoptosis （FITC Annexin V positive and PI negative）. A minor

population of cells were observed to be FITC Annexin V and PI

positive, indicating that they were in end stage apoptosis or already

dead.

Application Notes

Application

Flow cytometry Routinely Tested

Recommended Assay Procedure:

FITC Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces （Kd of ~5 x 10^-2） with

a higher affinity for phosphatidylserine （PS） than most other phospholipids. FITC Annexin V binding is calcium dependent and defined calcium

and salt concentrations are required for optimal staining as described in the FITC Annexin V Staining Protocol. Investigators should note that

FITC Annexin V flow cytometric analysis on adherent cell types （e.g HeLa, NIH 3T3, etc.） is not routinely tested as specific membrane

damage may occur during cell detachment or harvesting. Methods for utilizing Annexin V for flow cytometry on adherent cell types,

however, have been previously reported （Casiola-Rosen et al. and van Engelend et al.）.

INDUCTION OF APOPTOSIS BY CAMPTOTHECIN

The following protocol is provided as an illustration on how FITC Annexin V may be used on a cell line （Jurkat）.

Materials

1. Prepare Camptothecin stock solution （Sigma-Aldrich Cat. No. C-9911）: 1 mM in DMSO.

2. Jurkat T cells （ATCC TIB-152）.

Procedure

1. Add Camptothecin （final conc. 4-6 μM） to 1 x 10^6 Jurkat cells.

2. Incubate the cells for 4-6 hr at 37°C.

3. Proceed with the FITC Annexin V Staining Protocol to measure apoptosis.

FITC ANNEXIN V STAINING PROTOCOL

FITC Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on

the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid

phosphatidylserine （PS） is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external

environment. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying

apoptotic cells with exposed PS. Propidium Iodide （PI） is a standard flow cytometric viability probe and is used to distinguish viable from

nonviable cells. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Cells that

stain positive for FITC Annexin V and negative for PI are undergoing apoptosis. Cells that stain positive for both FITC Annexin V and PI are

either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both FITC Annexin V and PI are

alive and not undergoing measurable apoptosis.

556547 Rev. 5 Page 2 of 3

Reagents

1. FITC Annexin V （component no. 51-65874X）: Use 5 μl per test.

2. Propidium Iodide （PI）（component no. 51-66211E） is a convenient, ready-to-use nucleic acid dye. Use 5 μl per test.

3. 10X Annexin V Binding Buffer （component no. 51-66121E）: 0.1 M Hepes/NaOH （pH 7.4）, 1.4 M NaCl, 25 mM CaCl2. For a 1X working

solution, dilute 1 part of the 10X Annexin V Binding Buffer to 9 parts of distilled water.

Staining

1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10^6 cells/ml.

2. Transfer 100 μl of the solution （1 x 10^5 cells） to a 5 ml culture tube.

3. Add 5 μl of FITC Annexin V and 5 μl PI.

4. Gently vortex the cells and incubate for 15 min at RT （25°C） in the dark.

5. Add 400 μl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.

SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY

The following controls are used to set up compensation and quadrants:

1. Unstained cells.

2. Cells stained with FITC Annexin V （no PI）.

3. Cells stained with PI （no FITC Annexin V）.

Other Staining Controls:

A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with FITC Annexin V and/or FITC

Annexin V and PI. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in the

absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis （FITC Annexin V

positive, PI negative or FITC Annexin V positive, PI positive）.

The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo

apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from percentage of apoptotic cells in the

treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged

membrane and stain positive for PI as well as for FITC Annexin V. Thus the assay does not distinguish between cells that have already undergone

an apoptotic cell death and those that have died as a result of necrotic pathway, because in either case the dead cells will stain with both FITC

Annexin V and PI.

Product Notices

1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.