{"product_id":"biolegend-640922","title":"Biolegend, 640922, FITC Annexin V Apoptosis Detection Kit with 7-AAD, 100tests","description":"\u003cp\u003eBioLegend's FITC Annexin V Apoptosis Detection Kit with 7-AAD has been specifically designed for the identification of apoptotic and necrotic cells.  Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. Annexin V Binding Buffer is recommended for use with Annexin V staining. Annexin V binding alone cannot differentiate between apoptotic and necrotic cells. To help distinguish between the necrotic and apoptotic cells we recommend use of our 7-amino-actinomycin D (7-AAD) solution. Early apoptotic cells will exclude 7-AAD, while late stage apoptotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA.  7-AAD (7-amino-actinomycin D) has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis. When excited by 488 laser light, 7-AAD fluorescence is detected in the far red range of the spectrum (650 nm long-pass filter). \u003cbr\u003e\n100tests\u003cbr\u003e\nVerified Reactivity: Human, Mouse, Rat\u003cbr\u003e\nReported Reactivity: Other Species\u003cbr\u003e\nConcentration: Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)\u003cbr\u003e\nStorage \u0026amp; Handling: Store between 2°C and 8°C. Do not freeze. Caution: 7-AAD is a potential carcinogen. It is recommended that the user wear protective clothing, gloves, and eye\/face protection in order to avoid contact with skin and eyes.\u003cbr\u003e\nApplication: FC - Quality tested\u003cbr\u003e\nExcitation Laser: Blue Laser (488 nm)\u003cbr\u003e\nApplication Notes: Annexin V Staining Wash cells twice with cold BioLegend Cell Staining Buffer (Cat. No. 420201) and then resuspend the desired amount of cells in Annexin V Binding Buffer (Cat. No. 422201) at a concentration of 0.25-1.0 x 107 cells\/mL Transfer 100 µL of cell suspension in 5 mL test tube. Add 5 µL of fluorochrome conjugated Annexin V. Stain with a viability dye, such as PI (Cat. No. 421301), 7-AAD (Cat. Nos. 420403 \u0026amp; 420404), or Helix NP dyes (Cat. Nos. 425301, 425303, \u0026amp; 425305), if desired. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark. Add 400* µL of Annexin V Binding Buffer (Cat. No. 422201) to each tube. *For more concentrated samples, add a minimum of 200 µl of Annexin V Binding Buffer in this step. Analyze by flow cytometry. For a better experience detecting apoptosis, we now recommend Apotracker™. Cell staining with Apotracker™ is Calcium independent. Thus, no special buffers are required, and the protocol can be shortened for single-step co-staining with other reagents.\u003cbr\u003e\nApplication References(PubMed link indicates BioLegend citation): Maciel E, et al. 2014. Arch Biochem Biophys. 548:38. PubMed\u003cbr\u003e\nProduct Citations: Ferriz M, et al. 2023. STAR Protoc. 4:102079. PubMed Barrios-Bernal P, et al. 2023. Int J Mol Sci. 24:. PubMed Ribeiro MS, et al. 2023. Methods Mol Biol. 2618:289. PubMed Mohammadpour H, et al. 2023. Cell Rep. 42:112250. PubMed Park SA, et al. 2023. Int J Mol Sci. 24:. PubMed Groß E, et al. 2023. Cancers (Basel). 15:. PubMed Delangre E, et al. 2021. Cell Death Dis. 12:1136. PubMed Zhu Y, et al. 2022. Oxid Med Cell Longev. 2022:3182931. PubMed Dubiella C, et al. 2021. Nat Chem Biol. 17:954. PubMed Silveira DB, et al. 2022. Cell Biochem Funct. 40:163. PubMed Jang HJ, et al. 2022. Cancer Res. 82:2593. PubMed Bellanti F, et al. 2023. Int J Mol Sci. 24: . PubMed Lee H, et al. 2019. Cancer Res. 79:2839. PubMed Staresinic B, et al. 2018. Sci Rep. 8:9412. PubMed Witney T, et al. 2015. Clin Cancer Res. 21: 3896-3905. PubMed Maeda J, et al. 2016. PLoS One. 11: 0156689. PubMed Wang Z, et al. 2020. Cancer Manag Res. 3.563194444. PubMed Deng H, et al. 2020. Nat Commun. 3.896527778. PubMed Sima LE, et al. 2021. J Immunother Cancer. 9:. PubMed Khodeer S, et al. 2022. J Cell Sci. 135: . PubMed Karabicici M, et al. 2021. Mol Oncol. 15:2185. PubMed Liu M, et al. 2016. Cell Mol Gastroenterol Hepatol. 2:63-76. PubMed Demelash A, et al. 2015. J Biol Chem. 290: 21962-21975. PubMed Saunders IT, et al. 2019. Cancer Cell Int. 19:98. PubMed Zhang Q, et al. 2021. Theranostics. 11:8301. PubMed Koolivand M, et al. 2022. Cell J. 24:44. PubMed Znidar K, Bosnjak M 2015. Mol Ther Nucleic Acids. 5:e322. PubMed Tsuchiya N, et al. 2020. Cell Reports. 29(1):162-175.e9.. PubMed Zietzer A, et al. 2020. J Extracell Vesicles. 9:1786967. PubMed Khan H, et al. 2019. Cell Rep. 29:3009. PubMed Ma L, et al. 2015. Blood. 126: 247 - 256. PubMed Martínez-Montemayor MM, et al. 2019. Front Pharmacol. 10:115. PubMed Davies SP, et al. 2019. Cell Rep. 29:1610. PubMed Davenne T, et al. 2020. Cell Rep. 31:107640. PubMed Ruivo CF, et al. 2022. Gut. . PubMed Ma J, et al. 2021. Curr Protoc. 1:e144. PubMed Yan C, et al. 2019. Cancer Biol Med. 16:109. PubMed Oshima R, et al. 2016. Sci Rep. 6: 24997. PubMed Zhang H, et al. 2020. Proc Natl Acad Sci U S A. 117:29862. PubMed Wang L, et al. 2022. Am J Transl Res. 14:150. PubMed Yang Q, et al. 2016. Mol Biol Cell. 27: 197 - 208. PubMed Lee HS, et al. 2019. Interact Cardiovasc Thorac Surg. 28:353. PubMed Kim S, et al. 2021. J Clin Invest. 131:. PubMed Maciel E, et al. 2014. Arch Biochem Biophys. 548:34. PubMed Ito T, et al. 2021. Commun Biol. 4:694. PubMed Zhang D, et al. 2020. Stem Cell Res Ther. 0.816666667. PubMed Chen Z, et al. 2019. J Exp Med. 216:152. PubMed Parv K, et al. 2021. Front Endocrinol (Lausanne). 12:606175. PubMed Boemi I, et al. 2021. Front Cell Dev Biol. 9:555248. PubMed Gudikote JP, et al. 2021. J Biol Chem. 297:101163. PubMed Perkhofer L, et al. 2016. Stem Cell Res. 17: 367-378. PubMed Osei-Amponsa V, et al. 2020. Mol Cell Biol. 40:00:00. PubMed Chen SM, et al. 2021. Biomolecules. 11:. PubMed Ma S, et al. 2016. Cell Death Dis. 7:e2307. PubMed Wright JA, et al. 2021. Front Immunol. 12:705307. PubMed Karabicici M, et al. 2021. Front Cell Dev Biol. 9:639779. PubMed Bianchi M, et al. 2021. Innate Immun. 27:89. PubMed Pan Y, et al. 2017. Nature. 543:252-256. PubMed Sim CK, et al. 2019. J Immunol Res. 2019:7596786. PubMed Lang L, et al. 2017. J Exp Clin Cancer Res. 10.1186\/s13046-017-0583-4. PubMed Krohn S, et al. 2022. Front Immunol. 13:908093. PubMed Pham DV, et al. 2022. J Exp Clin Cancer Res. 41:9. PubMed Zietzer A, et al. 2021. Cell Mol Life Sci. 79:48. PubMed Wei L, et al. 2015. PLoS One. 10: 0131763. PubMed Artru F, et al. 2020. J Hepatol. 72:1052. PubMed Urdinguio RG, et al. 2019. Nucleic Acids Res. 47:5016. PubMed Yang L, et al. 2021. Front Immunol. 12:722273. PubMed Simula L, et al. 2020. Cell Death Differ. 27:2749. PubMed Barbu EA, et al. 2020. Front Immunol. 1.385416667. PubMed Notara M, et al. 2015. Stem Cell Res. 15: 643-654. PubMed Aggarwal N, et al. 2021. Cell Rep. 37:110170. PubMed Balani DH, et al. 2021. J Bone Miner Res. 36:757. PubMed\u003cbr\u003e\nBiology Area: Apoptosis\/Tumor Suppressors\/Cell Death, Cell Biology, Cell Proliferation and Viability, Neuroscience\u003cbr\u003e\nMolecular Family: Cytokines\/Chemokines\u003cbr\u003e\nGene ID: 308\u003cbr\u003e\nRegulatory Status: RUO\u003cbr\u003e\nOther Names: Annexin A5 Apoptosis Detection Kit, Dead, Apoptosis\u003cbr\u003e\nQ: How is your Annexin made and what sequence does it cover?\u003cbr\u003e\nA: It is made in E. coli, covering human aa Met1-Asp320.\u003cbr\u003e\nQ: How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?\u003cbr\u003e\nA: Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.\u003cbr\u003e\nQ: Why do I need to use Annexin V Binding Buffer?\u003cbr\u003e\nA: Annexin V binding requires the presence of calcium in the solution.  So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.\u003cbr\u003e\nQ: Why is washing not recommended after the addition 7-AAD or PI addition when assessing viability?\u003cbr\u003e\nA: These dyes bind in equilibrium with DNA. Therefore, external dye concentration must be maintained during analysis and the dye should not be washed out.\u003cbr\u003e\nQ: Can I use RPMI during Annexin V staining?\u003cbr\u003e\nA: It is best to follow protocol as described on the product data sheet. Moreover, RPMI 1640 has a relatively high concentration of phosphate and low calcium ion concentration, which negatively impacts Annexin binding to its target phosphatidylserine (PS). Measurement of cell death by using Annexin V may also be significantly affected by time of incubation on ice, calcium concentration, and type of medium.\u003cbr\u003e\nQ: Can I freeze Annexin V conjugates?\u003cbr\u003e\nA: It should not be frozen as it will lead to loss of biological activity due to dimerization.\u003cbr\u003e\nQ: Is Annexin V suitable for conjugation with the Maxpar® kit for CyTOF®?\u003cbr\u003e\nA: Maxpar® Labeling kits require the protein to be partially reduced, so the metal chelate can be introduced through an SH group in the hinge region of the reduced antibody. Human Annexin V contains only one Cysteine which was reported to be chemically inactive. Thus, the Maxpar® labeling protocol would not work with Annexin V, unless a free –SH group can be introduced to Annexin V.  For more information regarding SH-mediated conjugation of Annexin V please consult published papers such as this one.\u003c\/p\u003e","brand":"Biolegend","offers":[{"title":"Default Title","offer_id":46864300703913,"sku":"640922","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/biolegend-640922","provider":"Iright","version":"1.0","type":"link"}