Biolegend, 640920, APC Annexin V, 100tests

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 (Cat. No. 422201) is recommended for use with Annexin V staining. Annexin V binding alone cannot differentiate between apoptotic cells and necrotic. Therefore, we recommend using our Helix NP™ Blue (Cat No. 425305), Helix NP™ Green (Cat No. 425303) or Helix NP™ NIR (Cat. No. 425301). Early apoptotic cells will exclude 7-AAD and 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.
100tests
Verified Reactivity: Human, Mouse, Rat
Reported Reactivity: Other Species
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA)
Preparation: The purified protein was conjugated with APC under optimal conditions.
Concentration: Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling: The Annexin V solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: FC - Quality tested
Recommended Usage: Each lot of this product is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per 100,000 - million cells in a 100 µl volume of Annexin V Binding Buffer (Cat No. 422201). It is recommended that the reagent be titrated for optimal performance for each application.
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 cells in Annexin V Binding Buffer (Cat. No. 422201) at a concentration of 1x106 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): Koopman G, et al. 1994. Blood 84:1415. Vermes I, et al. 1995. J. Immunol. Methods 184:39. Dachary-Prigent J, et al. 1993. Blood 81:2554. Sekine C, et al. 2009. Int Immunol. PubMed Grujic M, et al. 2010. J. Immunol. 185:1730. PubMed Hussain MS, et al. 2013. Hum Mol Genet. 22:5199. PubMed Feng Q, et al. 2014. PLoS One. 9:95927. PubMed Isobe T, et al. 2014. eLife. 3:1977. PubMed
Product Citations: Isobe T, et al. 2014. Elife. 3:1977. PubMed Tang Z, et al. 2017. Cell Death Differ. 24:2127. PubMed Patel RP, et al. 2020. Transl Oncol. 14:100917. PubMed Kalus P, et al. 2022. Int J Mol Sci. 23:. PubMed McCartin C, et al. 2022. Chem Biol Interact. 367:110167. PubMed Hagege A, et al. 2022. Cell Rep Med. 3:100659. PubMed Christian DA, et al. 2022. Sci Immunol. 7:eabq7432. PubMed Shiroshita K, et al. 2022. Cell Rep Methods. 2:100354. PubMed Giampaolo S, et al. 2023. iScience. 26:106234. PubMed Battistello E, et al. 2023. Mol Cell. 83:1216. PubMed Riera-Domingo C, et al. 2023. Sci Adv. 9:eadd5028. PubMed Perez GI, et al. 2023. Vaccines (Basel). 11:. PubMed Chatterjee S, et al. 2023. Sci Adv. 9:eade4800. PubMed Onyshchenko K, et al. 2023. Nat Commun. 14:2087. PubMed Pang JM, et al. 2023. PLoS One. 18:e0284744. PubMed Van Bael J, et al. 2023. Int J Mol Sci. 24:. PubMed Xiao C, et al. 2023. Nat Aging. 3:418. PubMed Sun K, et al. 2023. Cancers (Basel). 15:. PubMed Wyrobnik I, et al. 2023. Oncoimmunology. 12:2219164. PubMed Jiang D, et al. 2023. Nat Commun. 14:3642. PubMed Preda MB, et al. 2021. Cell Death Dis. 12:566. PubMed Chugh RM, et al. 2021. Stem Cell Res Ther. 12:388. PubMed Inclan-Rico JM, et al. 2020. Nat Immunol. 21:1181. PubMed Sugiura A, et al. 2022. Immunity. 55:65. PubMed Zhang H, et al. 2022. Eur J Immunol. 52:978. PubMed Hao J, et al. 2022. Cell Rep. 41:111804. PubMed Cappellesso F, et al. 2022. Nat Cancer. 3:1464. PubMed Liu X, et al. 2023. Nat Commun. 14:238. PubMed Di Benedetto C, et al. 2023. Cancers (Basel). 15: . PubMed Daian LM, et al. 2023. Int J Mol Sci. 24: . PubMed Hochrein SM, et al. 2022. Cell Metab. 34:516. PubMed Roy S, et al. 2022. Endocrinology. 163:. PubMed McCartin C, et al. 2022. Cancers (Basel). 14:. PubMed Xu B, et al. 2017. Oncogenesis. 6:e295. PubMed Wu N, et al. 2020. Cell Rep. 30:1129. PubMed Wiley CD, et al. 2019. Cell Rep. 28:3329. PubMed Kuljanin M, et al. 2018. Cell Rep. 25:2524. PubMed Wu SJ, et al. 2017. Cell Rep. 2.628472222. PubMed Schick M, et al. 2022. Nat Commun. 13:281. PubMed Dai X, et al. 2017. J Cell Mol Med. 10.1111/jcmm.13296. PubMed Donado CA, et al. 2020. Cell Reports. 31(1):107466. PubMed Cannons JL, et al. 2021. Cell Rep. 37:109804. PubMed Zeriouh W, et al. 2017. PLoS One. 12(2):e0170823. PubMed Weiser J, et al. 2015. J Infect Dis. 212: 1677 - 1682. PubMed Zhang S, et al. 2019. Front Pediatr. 0.485416667. PubMed Vig S, et al. 2019. Cell Death Dis. 0.63125. PubMed Xiao C, et al. 2022. iScience. 25:103934. PubMed McNeal AS, et al. 2021. Elife. 10:. PubMed Wang J, et al. 2016. Nat Cell Biol. 18: 480-490. PubMed Sumitomo Y, et al. 2016. Blood. 128: 1614 - 1624. PubMed Li L, et al. 2016. Cell Death Dis. 7:e2439. PubMed Wohlhieter CA, et al. 2020. Cell Rep. 33:108444. PubMed Pease NA, et al. 2021. Cell Reports. 34(12):108888. PubMed Cao J, et al. 2019. Theranostics. 0.938194444. PubMed Moose DL, et al. 2020. Cell Rep. 30:3864. PubMed Figueroa-Valdés AI, et al. 2021. Front Bioeng Biotechnol. 9:619930. PubMed Fujino T, et al. 2021. Nat Commun. 12:1826. PubMed Patel RP, et al. 2021. Invest Ophthalmol Vis Sci. 62:16. PubMed Dregalla RC, et al. 2021. Stem Cell Res Ther. 12:547. PubMed Linnerbauer M, et al. 2022. Front Immunol. 12:800128. PubMed Vongpipatana T, et al. 2020. J Immunol. 2156:204. PubMed O'Neill K, et al. 2016. Genes Dev. 30: 973-988. PubMed Hussain M, et al. 2013. Hum Mol Genet. 22:5199. PubMed Zeng H, et al. 2018. Cancer Cell. 34:56:00. PubMed Lei W, et al. 2020. Sci Rep. 10:7004. PubMed Ning X, et al. 2019. Mol Cell. 74:19. PubMed Deng J, et al. 2021. Cell Death Dis. 12:978. PubMed Hickman O, et al. 2016. Br J Cancer. 10.1038/bjc.2016.91. PubMed Laughney AM, et al. 2020. Nat Med. 26:259. PubMed Hagan AS, et al. 2020. Development. 147:00:00. PubMed Sheng Y, et al. 2019. Cell Prolif. 52:e12611. PubMed Tang Z, et al. 2019. Cell Rep. 28:1744. PubMed Liu L, et al. 2019. Cell Prolif. :e12718. PubMed Fleischmann M, et al. 2021. Cells. 10:. PubMed Rausch M, et al. 2021. Cancers (Basel). 13:. PubMed Lee M, et al. 2022. Nat Commun. 13:1157. PubMed Høgh RI, et al. 2020. J Immunol. 1746:204. PubMed Zhang S, et al. 2020. Front Immunol. 1.757638889. PubMed Saint Fleur–Lominy S, et al. 2018. Cell Rep. 24:3045. PubMed Kanda K, et al. 2018. JCI Insight. 3:e91316. PubMed Zhou X, et al. 2019. Cell Rep. 27:1176. PubMed Lee Y, et al. 2022. Sci Adv. 8:eabm7688. PubMed Qiu C, et al. 2022. Front Immunol. 12:764949. PubMed Aslam MA, et al. 2021. EMBO Rep. 22:e51184. PubMed Cao W, et al. 2016. Nat Commun. 7:11687. PubMed Perny M, et al. 2016. Cell Death Dis. 7:e2447. PubMed Liu L, et al. 2020. Cancer Res. 80:2564. PubMed Brasacchio D, et al. 2018. Cell Death Dis. 0.681944444. PubMed Celus W, et al. 2021. Cancer Immunol Res. Online ahead of print. PubMed Cai J, et al. 2021. eLife. 10:00. PubMed Cheng CC, et al. 2020. Elife. 9:00. PubMed Feng Q, et al. 2014. PLoS One. 9:95927. PubMed Jung E, et al. 2017. J Neurosci. 37:6837. PubMed Ren Y, et al. 2022. J Immunother Cancer. 10:. PubMed Warmuth S, et al. 2022. Oncoimmunology. 10:2004661. PubMed Hills LB, et al. 2021. J Immunol. 206:89. PubMed Dou R, et al. 2021. Cell Death Dis. 12:206. PubMed Burclaff J, et al. 2022. Cell Mol Gastroenterol Hepatol. 13:1554. PubMed Pedicona F, et al. 2022. Sci Signal. 15:eabl7989. PubMed Li R, et al. 2022. Exp Hematol Oncol. 11:77. PubMed He C, et al. 2022. BMC Cancer. 22:1100. PubMed Li C, et al. 2021. Cell Metabolism. 33(8):1610-1623.e5. PubMed Kostadinova E, et al. 2016. Sci Rep. 6:30943. PubMed Qiu Z, et al. 2019. Theranostics. 3.650694444. PubMed Reyes–Uribe P, et al. 2018. Oncogene. 37:4058. PubMed Lima S, et al. 2018. Autophagy. 1.2375. PubMed Oh J, et al. 2017. PLoS Biol. 15:e2003352. PubMed Lasrado N, et al. 2022. iScience. 25:103865. PubMed Forsberg MH, et al. 2021. Stem Cell Res Ther. 12:459. PubMed Okita R, et al. 2021. Thorac Cancer. 12:775. PubMed Fernández D, et al. 2016. PLoS One. 11: 0157889. PubMed Muthalagu N, et al. 2020. Cancer Discov. 1.022222222. PubMed Pothlichet J, et al. 2020. J Clin Invest. 130:2872. PubMed Wei Z, et al. 2021. Nat Commun. 0.805555556. PubMed Sun J et al. 2018. Cell stem cell. 23(3):355-369 . PubMed Sharma P, et al. 2018. Data Brief. 1.975694444. PubMed Li B, et al. 2019. Oncol Rep. 41:608. PubMed Millington-Burgess SL, et al. 2021. Sci Rep. 11:17678. PubMed Sahoo SS, et al. 2021. Nat Med. 27:1806. PubMed Hirata SI, et al. 2020. Allergy. 75:1939. PubMed Vickman R, et al. 2016. Mol Cancer Res. 14: 776 - 786. PubMed Cabet E, et al. 2015. PLoS One. 10: 0137009. PubMed Burgener SS, et al. 2019. Cell Rep. 27:3646. PubMed Hasselluhn MC, et al. 2019. Cell Death Dis. 0.726388889. PubMed Zhang S, et al. 2018. Sci Rep. 8:17066. PubMed Shinde R, et al. 2018. Nat Immunol. 1.188194444. PubMed Rodriguez-Perdigon M, et al. 2022. Macromol Biosci. 22:e2200168. PubMed Xu Y, et al. 2021. IUBMB Life. 73:1432. PubMed Ho JY, et al. 2021. Mol Ther Methods Clin Dev. 21:237. PubMed Yamamoto H, et al. 2016. Sci Rep. 6:19204. PubMed Dong G, et al. 2020. Haematologica. . PubMed Alamillo E, et al. 2017. Fish Shellfish Immunol. 10.1016/j.fsi.2017.08.036. PubMed Voss OH, et al. 2018. Curr Protoc Immunol. 120:14.44.1. PubMed Dower CM, et al. 2018. Mol Cancer Ther. 2.350694444. PubMed Barve A, et al. 2019. Cells. 1.251388889. PubMed Tulasi DY, et al. 2021. Cell Mol Gastroenterol Hepatol. 11:1437. PubMed Satcher R, et al. 2015. J Cell Sci. 128: 4629 - 4641. PubMed Grossman L, et al. 2017. mSphere. 2:e00305. PubMed Habtetsion T et al. 2018. Cell metabolism. 28(2):228-242 . PubMed Woolf N, et al. 2017. Oncogenesis. 10.1038/oncsis.2017.77. PubMed Parsa S, et al. 2021. Nat Cancer. 1:653. PubMed Li S, et al. 2021. Front Pharmacol. 12:634115. PubMed Höhne K, et al. 2016. Open Bio. 6: 160046. PubMed Festag J, et al. 2020. Mol Ther Nucleic Acids. 1.330555556. PubMed Wallace JG, et al. 2020. Clin Immunol. 210:108311. PubMed Cordo' V, et al. 2022. Nat Commun. 13:1048. PubMed Mykhailova O, et al. 2021. Transfusion. 61:1247. PubMed Walle T, et al. 2022. Sci Adv. 8:eabh4050. PubMed Loo Yau H, et al. 2021. Molecular Cell. 81(7):1469-1483.e8. PubMed Viviane Ponath, Bernd Kaina 2017. PLoS One. 10.1371/journal.pone.0170347. PubMed Yang X, et al. 2020. Immunity. 51(6):983-996.e6.. PubMed Chen Y, et al. 2020. Front Oncol. 10:767. PubMed Lu Y, et al. 2018. Cancer Cell. 33:1048. PubMed Agarwal P, et al. 2021. Cell Reports. 36(2):109386. PubMed Song S, et al. 2021. Mar Drugs. 19: . PubMed Schäfer AL, et al. 2021. Front Immunol. 12:696810. PubMed Anderson CJ, et al. 2021. Nature. 596:262. PubMed Trigg RM, et al. 2019. Nat Commun. 4.186111111. PubMed Young MM, et al. 2019. Cell Death Dis. 1.004861111. PubMed Chen Y, et al. 2022. Cell Death Dis. 13:822. PubMed Amend A, et al. 2021. Int J Mol Sci. 22:. PubMed
RRID: not an antibody (BioLegend Cat. No. 640919) AB_2561515 (BioLegend Cat. No. 640920) AB_2616657 (BioLegend Cat. No. 640941)
Biology Area: Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Neuroscience
Gene ID: 308
UniProt: View information about Annexin V on UniProt.org
Regulatory Status: RUO
Other Names: Annexin A5, 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.