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产品名称:细胞凋亡试剂盒FITC Annexin V Apoptosis Detection Kit I

产品型号:BD 556547

产品报价:

产品特点:NameAnnexin V : FITC Apoptosis Detection Kit IContentsAnnexin V-FITC, Propidium Iodide Staining Solution, Annexin V Binding BufferSize100 TestsRegulatory Status RUO

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BD 556547细胞凋亡试剂盒FITC Annexin V Apoptosis Detection Kit I的详细资料:

Technical Data Sheet

澳门百家必胜方法FITC Annexin V Apoptosis Detection Kit I

Product Information

Material Number: 556547

澳门百家必胜方法Component: 51-66121E

Description: 10X Annexin V Binding Buffer

Size: 50 ml (1 ea)

澳门百家必胜方法Storage Buffer: Aqueous buffered solution containing no preservative.

Component: 51-65874X

澳门百家必胜方法Description: FITC Annexin V

Size: 0.5 ml (1 ea)

Vol. per Test: 5 μl

澳门百家必胜方法Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.

Component: 51-66211E

澳门百家必胜方法Description: Propidium Iodide Staining Solution

Size: 2.0 ml (1 ea)

Vol. per Test: 5 μl

Storage Buffer: Aqueous buffered solution containing no preservative.

Description

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

澳门百家必胜方法apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment,

澳门百家必胜方法condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features.

澳门百家必胜方法In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma

澳门百家必胜方法membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding

澳门百家必胜方法protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including FITC.

澳门百家必胜方法This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing

澳门百家必胜方法apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, FITC Annexin V staining can identify apoptosis at an earlier

澳门百家必胜方法stage than assays based on nuclear changes such as DNA fragmentation.

澳门百家必胜方法FITC Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either

澳门百家必胜方法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

澳门百家必胜方法positive). Viable cells with intact membranes exclude PI, wheras the membranes of dead and damaged cells are permeable to PI. For example,

澳门百家必胜方法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

澳门百家必胜方法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

澳门百家必胜方法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.

澳门百家必胜方法556547 Rev. 5 Page 1 of 3

澳门百家必胜方法Flow Cytometric Analysis of FITC Annexin V staining. Jurkat cells

澳门百家必胜方法(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

澳门百家必胜方法containing propidium iodide (PI) and analyzed by flow cytometry.

澳门百家必胜方法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.

澳门百家必胜方法Application Notes

澳门百家必胜方法Application

Flow cytometry Routinely Tested

澳门百家必胜方法Recommended Assay Procedure:

澳门百家必胜方法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

澳门百家必胜方法and salt concentrations are required for optimal staining as described in the FITC Annexin V Staining Protocol. Investigators should note that

澳门百家必胜方法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.).

澳门百家必胜方法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

澳门百家必胜方法1. Prepare Camptothecin stock solution (Sigma-Aldrich Cat. No. C-9911): 1 mM in DMSO.

澳门百家必胜方法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.

澳门百家必胜方法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

澳门百家必胜方法phosphatidylserine (PS) is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external

澳门百家必胜方法environment. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying

澳门百家必胜方法apoptotic cells with exposed PS. Propidium Iodide (PI) is a standard flow cytometric viability probe and is used to distinguish viable from

澳门百家必胜方法nonviable cells. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Cells that

澳门百家必胜方法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

澳门百家必胜方法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.

澳门百家必胜方法2. Propidium Iodide (PI) (component no. 51-66211E) is a convenient, ready-to-use nucleic acid dye. Use 5 μl per test.

澳门百家必胜方法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

澳门百家必胜方法solution, dilute 1 part of the 10X Annexin V Binding Buffer to 9 parts of distilled water.

Staining

澳门百家必胜方法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.

澳门百家必胜方法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.

澳门百家必胜方法4. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.

澳门百家必胜方法5. Add 400 μl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.

澳门百家必胜方法SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY

澳门百家必胜方法The following controls are used to set up compensation and quadrants:

1. Unstained cells.

澳门百家必胜方法2. Cells stained with FITC Annexin V (no PI).

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

Other Staining Controls:

澳门百家必胜方法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

澳门百家必胜方法absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis (FITC Annexin V

澳门百家必胜方法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

澳门百家必胜方法apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from percentage of apoptotic cells in the

澳门百家必胜方法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

澳门百家必胜方法Annexin V and PI.

Product Notices

澳门百家必胜方法1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.

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

澳门百家必胜方法Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before

澳门百家必胜方法discarding to avoid accumulation of potentially explosive deposits in plumbing.

3.

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

References

澳门百家必胜方法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)

澳门百家必胜方法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)

澳门百家必胜方法O'Brien MC, Bolton WE. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow

澳门百家必胜方法cytometry. Cytometry. 1995; 19(3):243-255. (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)

澳门百家必胜方法Schmid I, Krall WJ, Uittenbogaart CH, Braun J, Giorgi JV. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence

澳门百家必胜方法in single laser flow cytometry. Cytometry. 1992; 13(2):204-208. (Biology)

澳门百家必胜方法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)

澳门百家必胜方法556547 Rev. 5 Page 3 of 3

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