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Annexin V-Super Fluor 647/PI 细胞凋亡检测试剂盒
Annexin V-Super Fluor 647/PI Dead Cell Apoptosis Detection Kit
Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. 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. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment.
Annexin V was found to play a major role in matrix vesicle-initiated cartilage calcification as a collage-regulated calcium channel. 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. 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.
Alexa Fluor 647 dye is a superior far-red-fluorescent dye with spectra similar to the Cy5 dye.The Alexa Fluor 647 dye, to provide the maximum sensitivity. Staining with Alexa Fluor 647 Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) to allow the investigator to identify early apoptotic cells (PI negative, Alexa Fluor 647 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 Alexa Fluor 647 Annexin V and PI negative; cells that are in early apoptosis are Alexa Fluor 647 Annexin V positive and PI negative; and cells that are in late apoptosis or already dead are are both Alexa Fluor 647 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 Alexa Fluor 647 Annexin V and PI. However, when apoptosis is measured over time, cells can be often tracked from Alexa Fluor 647 Annexin V and PI negative (viable, or no measurable apoptosis), to Alexa Fluor 647 Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to Alexa Fluor 647 Annexin V and PI positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis.
Contents and Storage Information：
|
Components |
20 assays |
50 assays |
100 assays |
Storage and Stability |
|
rh Annexin V-Super Fluor 647 |
100 μl |
250 μl |
500 μl |
4oC (Protect from light) |
|
Propidium Iodide, PI |
200 μl |
500 μl |
1000 μl |
4oC (Protect from light) |
|
Binding Buffer ( 4´ ) |
4 ml |
10 ml |
20 ml |
4oC |
Materials Required but Not Provided：
1. 5 ul to 1000 ul adjustable single channel micropipettes with disposable tips
2. Phosphate-buffered saline (PBS)
3. Glass-distilled or deionized water
4. Pancreatic enzyme can not contain EDTA
5. Bench top centrifuge
6. Flow Cytometer
Precaution for Use：
1. All regents are intended for reserch use only. Not for use in diagnostic or therapeutic procedures.
2. The trace reagent needs to centrifuge for several seconds to collect the reagent to the bottom of the tube and then open the cover for use.。
3. Propidium Iodide（PI）is a potential mutagen; avoid contact of skin or mucous membranes.
4. Do not mix or substitute reagents with those from other lots or other sources.
5. Do not use kit reagents beyond expiration date on label.
6. Do not expose kit reagents to strong light during storage or incubation.
7. This kit is used for flow cytometry and used to detect living cells, and the number of cells should not be less than 1x106.
8. After staining, it is advisable to detect as soon as possible, and the excessive time may lead to the increase of apoptosis or necrotic cells.
9. Annexin V's method of detecting apoptotic cells is suitable for the detection of cells with suspended growth, such as lymphocytes. For the growth of cell wall, such as in pancreatic enzyme digestion process can cause cell membrane damage, which will lead to high false positive. The cells digested by pancreatic enzyme can be stored in PBS containing 2% BSA.
10. Annexin V-Alexa Fluor 488 can be degraded by the residual pancreatic enzymes, resulting in staining failure.
11. Cell fixation may cause fluorescence quenching, please do not fix cells.。
Preparation of Reagents:
Dilute Binding Buffer (4X) 1:4 in distilled/deionized water (20ml binding buffer and 60 ml distilled/deionized water).
Experimental Procedure：
1. Preparation of cell samples：
a) Suspension cells：
i. Harvest the cells to centrifuge tube and be centrifuged at 1000-2000rpm for 5 minute, and discard the supernatant.
ii. Wash cells in 1ml cold phosphate-buffered saline (PBS) by gentle shaking or pipetting up and down, and counting cells.
iii. Cells are centrifuged at 1000-2000rpm for 5 minute, and discard the supernatant.
iv. Repeat steps ii and iii.
b) Adherent cells：
i. Inhaled the cell culture medium to the centrifuge tube, and then wash adherent cells in phosphate-buffered saline (PBS),
ii. Add pancreatic enzyme that can not contain EDTA to digest the adherent cells.
iii. Incubate at room temperature untill the cells are blown down by a gentle blow. To avoid excessive digestion..
iv. Harvest digested cells into the centrifuge tube in step i.
v. Cells are centrifuged at 1000-2000rpm for 5 minute, and discard the supernatant.
vi. Wash cells in 1ml cold phosphate-buffered saline (PBS) by gentle shaking or pipetting up and down, and counting cells.
vii. Cells are centrifuged at 1000-2000rpm for 5 minute, and discard the supernatant.
viii. Repeat steps vi and vii.
2. Resuspend the cells in 250 ul 1x Binding Buffer at a concentration of 1x 106 cells/ml.
3. Remove 100 ul cell suspension in 5ml flow tube, add 5ul Annexin V-Alexa Fluor 647, mix gently.
4. Incubate the cells at room temperature for 10min, protected from light..
5. Add 10 ul Propidium Iodide (PI) to the cell suspension before flow analysis, mix gently.
6. Add 400 ul 1X Binding Buffer to the cell suspension, mix gently, keep the samples on ice.
7. As soon as possible, analyze the stained cells by flow cytometry.
References：
1．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)
2．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)
3．Koopman G, Reutelingsperger 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)
4．Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger 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)
5．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)
6．Martin SJ, Reutelingsperger 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)
7．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)
8．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)
9．Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger 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)
NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.
Description
| Name | Annexin V-Super Fluor 647/PI Dead Cell Apoptosis Detection Kit | ||
|---|---|---|---|
| CAT# | 2022 | CAS# | |
| Storage# | -20 °C Sealed & desiccated & Minimized light exposure & Minimized Freezing And Thawing | Shelf Life# | 24 months |
| Ex(nm)# | 647 | Em(nm)# | 668 |
| MW# | Solvent# | DMSO | |
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