FITCAnnexinVApoptosisDetectionKitI
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BD Pharmingen?Technical Data Sheet
FITC Annexin V Apoptosis Detection Kit I
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Product Information
Material Number:556547
Component:51-66121E
10X Annexin V Binding Buffer
Description:
Size: 50 ml (1 ea)
Storage Buffer:Aqueous buffered solution containing no prervative.
Component:51-65874X
FITC Annexin V
Description:
Size: 0.5 ml (1 ea)
Vol. per Test: 5 µl
Storage Buffer:Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
Component:51-66211E
Propidium Iodide Staining Solution
Description:
Size: 2.0 ml (1 ea)
Vol. per Test: 5 µl
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Storage Buffer:Aqueous buffered solution containing no prervative.
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 phosphatidylrine (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 expod PS. Annexin V may be conjugated to fluorochromes including FITC.
This format retains its high affinity for PS and thus rves as a nsitive 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 bad 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 process. Therefore, staining with FITC Annexin V is typically ud 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 tho that have died as a result of a necrotic pathway becau in either ca, 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 prent) and finally to FITC Annexin V and PI positive (end stage apoptosis and death). The movement of cells through the three stages suggests apoptosis. In contrast, a single obrvation indicating that cells are both FITC Anne
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xin V and PI positive, in of itlf, reveals less information about the process by which the cells underwent their demi.
Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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 obrved 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 nsitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces (Kd of ~5 x 10^-2) with a higher affinity for phosphatidylrine (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-Ron 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 ud 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 ud to quantitatively determine the percentage of cells within a population that ar
e actively undergoing apoptosis. It relies on the property of cells to lo membrane asymmetry in the early phas of apoptosis. In apoptotic cells, the membrane phospholipid
phosphatidylrine (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 uful for identifying apoptotic cells with expod PS. Propidium Iodide (PI) is a standard flow cytometric viability probe and is ud 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.
Reagents
1. FITC Annexin V (component no. 51-65874X): U 5 µl per test.
2. Propidium Iodide (PI) (component no. 51-66211E) is a convenient, ready-to-u nucleic acid dye. U 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 ud to t 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 ud 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 abnce 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 ud 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 th
e 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 tho that have died as a result of necrotic pathway, becau in either ca 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.glass
Source of all rum proteins is from USDA inspected abattoirs located in the United States.
2.
3.
Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before
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discarding to avoid accumulation of potentially explosive deposits in plumbing.
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4.
Plea refer to /doc/dcb4671ac281e53a5802ffe8.html /pharmingen/protocols for technical protocols.
References
Andree HA, Reutelingsperger 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-Ron L, Ron A, Petri M, Schlisl 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)
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Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger CP, Roos D. Human neutrophils lo their surface Fc gamma RIII and acquire
Annexin V binding sites during apoptosis in vitro. Blood. 1995; 85(2):532-540. (Biology)
Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylrine expression on
B cells undergoing apoptosis. Blood. 1994; 84(5):1415-1420. (Biology)
priMartin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylrine 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)
O'Brien MC, Bolton WE. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry. 1995; 19(3):243-255. (Biology)
Raynal P, Pollard HB. Annexins: the problem of asssing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins.
Biochim Biophys Acta. 1994; 1197(1):63-93. (Biology)
Schmid I, Krall WJ, Uittenbogaart CH, Braun J, Giorgi JV. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence
in single lar flow cytometry. Cytometry. 1992; 13(2):204-208. (Biology)葫芦的英文
van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger 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, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylrine expression on early apoptotic cells using fluorescein labelled
Annexin V. J Immunol Methods. 1995; 184(1):39-51. (Biology)
