Biolegend, 640932, APC Annexin V Apoptosis Detection Kit with PI, 100tests

BioLegend's APC Annexin V Apoptosis Detection Kit with PI 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 Propidium Iodide Solution (PI). Early apoptotic cells will exclude PI, while late stage apoptotic cells and necrotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA. Propidium iodide is a fluorescent dye that binds to DNA. When excited by 488 nm laser light, it can be detected with in the PE/Texas Red® channel with a bandpass filter 610/10. It is commonly used in evaluation of cell viability or DNA content in cell cycle analysis by flow cytometry.
100tests
Verified Reactivity: Human, Mouse, Rat
Reported Reactivity: Other Species
Concentration: Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling: Store between 2°C and 8°C. Do not freeze.Caution: Propidium Iodide Solution is toxigenic and mutagenic; handle with care.
Application: FC - Quality tested
Excitation Laser: Red Laser (633 nm)
Application 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 & 420404), or Helix NP dyes (Cat. Nos. 425301, 425303, & 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.
Application References(PubMed link indicates BioLegend citation): Ranganathan P, et al. 2014. J AM Soc Nephrol. 25:239. PubMed Bernhart E, et al. 2014. Neuro Onco. 16:933. PubMed
Product Citations: Zhou L, et al. 2020. Oxid Med Cell Longev. 2685310:2020. PubMed Shrestha C,et al. 2017. PLoS One. 10.1371/journal.pone.0186169. PubMed Grubb T, et al. 2022. Clin Cancer Res. 28:4689. PubMed Lampron MC, et al. 2023. Cells. 12:. PubMed Lee J, et al. 2023. Front Cell Dev Biol. 11:1144110. PubMed Yakovlev VA, et al. 2023. Front Oncol. 13:1124147. PubMed Chaturvedi S, et al. 2023. Cell. 186:2036. PubMed Cui Y, et al. 2023. Research (Wash D C). 6:0162. PubMed Ke C, et al. 2022. Cell Biol Int. 46:579. PubMed Del Rosario RCH, et al. 2022. Nat Microbiol. 7:312. PubMed Pal D, et al. 2022. Cell Rep Med. 3:100717. PubMed Gutierrez WR, et al. 2022. JCI Insight. 7: . PubMed Li W, et al. 2023. Immunol Cell Biol. 101:78. PubMed Guo F, et al. 2023. Am J Reprod Immunol. 89:e13663. PubMed Gong Z, et al. 2023. Exp Hematol Oncol. 12:7. PubMed Gao LT, et al. 2023. Cell Mol Life Sci. 80:58. PubMed Li X, et al. 2023. J Transl Med. 21:121. PubMed Bikorimana JP, et al. 2022. iScience. 25:105537. PubMed Rodrigues M, et al. 2022. Biochem J. 479:1891. PubMed Chaturvedi S, et al. 2022. Cell. 185:2086. PubMed Baldominos P, et al. 2022. STAR Protoc. 3:101795. PubMed Wang H, et al. 2020. Onco Targets Ther. 13:3903. PubMed O'Connor CM, et al. 2022. Cancer Res. 82:721. PubMed Blackburn CMR, et al. 2021. Atherosclerosis. 330:76. PubMed Azad P, et al. 2022. Exp Mol Med. :. PubMed Vitali E, et al. 2020. Cancers (Basel). 12:00. PubMed Daza–Cajigal V, et al. 2019. Front Immunol. 10:2065. PubMed Cremasco F, et al. 2021. PLoS One. 16:e0241091. PubMed Wang F, et al. 2021. J Exp Clin Cancer Res. 40:259. PubMed Sun X, et al. 2022. JCI Insight. 7: . PubMed Lv ZY, et al. 2022. J Inflamm Res. 15:2819. PubMed Dichtl S, et al. 2021. Sci Adv. 7: . PubMed Cousin N, et al. 2021. Cancer Res. 81:4133. PubMed Liu K, et al. 2021. Front Cell Dev Biol. 9:678209. PubMed Zou J, et al. 2015. PLoS One. 10: 0136843. PubMed Portman N, et al. 2020. Breast Cancer Res. 0.977083333. PubMed Wang Z, et al. 2017. Elife. 6:e30590. PubMed Wang J, et al. 2021. Int J Mol Med. 47:643. PubMed Yetkin D, et al. 2021. EXCLI J. 20:1394. PubMed Takahashi H, et al. 2019. Sci Rep. 1.136111111. PubMed Daza-Cajigal V, et al. 2022. Front Immunol. 13:888427. PubMed Li T, et al. 2021. Front Cell Dev Biol. 9:599020. PubMed Qi T, et al. 2022. Invest Ophthalmol Vis Sci. 63:11. PubMed Zhang Y, et al. 2021. Cell Death Discov. 7:192. PubMed Gomzikova MO, et al. 2020. Sci Rep. 10:10740. PubMed Nassa G, et al. 2019. Sci Adv. 5:eaav5590. PubMed Karagül MI, et al. 2020. EXCLI J. 19:532. PubMed Liu C, et al. 2021. Immun Inflamm Dis. 9:1749. PubMed Bernhart E, et al. 2014. Neuro Oncology. 16:933. PubMed Deventhiran J, et al. 2016. J Virol. 90: 222 - 231. PubMed He L, et al. 2021. Front Physiol. 12:664222. PubMed Yan Y, et al. 2021. Br J Cancer. 125:101. PubMed Zhang N, et al. 2021. Atherosclerosis. 334:39. PubMed Quintanal-Villalonga A, et al. 2022. Cancer Res. 82:472. PubMed Li Y, et al. 2020. Nat Cancer. 1:882. PubMed Gong Y, et al. 2020. J Neuroinflammation. 0.845833333. PubMed Zhang S, et al. 2021. Cell Death Discov. 49:7. PubMed Xu P, et al. 2020. Cancer Immunol Res. 8:1193. PubMed Lyu X, et al. 2022. Discov Oncol. 13:30. PubMed Harro CM, et al. 2020. J Clin Invest. . PubMed Gantenbein N, et al. 2018. Eur J Cancer. 101:165. PubMed González-Rodríguez P, et al. 2022. Cell Death Dis. 13:953. PubMed Lohberger B, et al. 2016. PLoS One. 11:e0168193. PubMed Przystupski D, et al. 2019. Front Pharmacol. 10:851. PubMed Dudek M, et al. 2021. Nature. 592:444. PubMed Javellana M, et al. 2022. Cancer Res. 82:169. PubMed Wang YM, et al. 2022. Oncoimmunology. 11:2034257. PubMed Surya R, et al. 2016. Carcinogenesis. 37: 635 - 645. PubMed Hu X, et al. 2020. Neoplasia. 1.290972222. PubMed Pogozhykh D, et al. 2017. Stem Cell Res Ther. 10.1186/s13287-017-0512-7. PubMed Widodo N, et al. 2022. F1000Res. 11:169. PubMed He S, et al. 2022. Ren Fail. 44:171. PubMed Baxley RM, et al. 2021. Nat Commun. 1626:12. PubMed Schnoor B, et al. 2022. Front Bioeng Biotechnol. 10:941817. PubMed Morretton JP, et al. 2022. EMBO Mol Med. 14:e15670. PubMed
Biology Area: Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Cell Proliferation and Viability, Neuroscience
Gene ID: 308
Regulatory Status: RUO
Other Names: Annexin A5 Apoptosis Detection Kit, Dead, Apoptosis
Q: How is your Annexin made and what sequence does it cover?
A: It is made in E. coli, covering human aa Met1-Asp320.
Q: How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?
A: Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.
Q: Why do I need to use Annexin V Binding Buffer?
A: 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.
Q: Can I use RPMI during Annexin V staining?
A: 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.
Q: Can I freeze Annexin V conjugates?
A: It should not be frozen as it will lead to loss of biological activity due to dimerization.
Q: Is Annexin V suitable for conjugation with the Maxpar® kit for CyTOF®?
A: 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.