Biolegend, 640918, Pacific Blue™ 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 Pacific Blue™ 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. * Pacific Blue™ has a maximum emission of 455 nm when it is excited at 405 nm. Prior to using Pacific Blue™ conjugate for flow cytometric analysis, please verify your flow cytometer's capability of exciting and detecting the fluorochrome. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Violet Laser (405 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): Gordy C, et al. 2011 Blood 117:618. PubMed Jia W, et al. 2011. J. Immunol. 186:5313. PubMed Naegele M, et al. 2012. J. Neuroimmunol. 242:60. PubMed Jenke AC, et al. 2013. PLoS One. 8:e55636. PubMed Oh J, et al. 2013. J Exp Med. 210:1069. PubMed Hasan S, et al. 2013. Blood. 122:1464. PubMed Zhao BB, et al. 2013. PLoS One. 8:77008. PubMed Mihaly SR, et al. 2014. PLoS One. 9:94982. PubMed Miyazawa M, et al. 2014. Mol Biol Cell. 25:2116. PubMed Yabas M, et al. 2014. J Biol Chem. 289:19531. PubMed Burbulla LF, et al. 2014. Cell Death Dis. 5:1180. PubMed
Product Citations: Tsuji M 2014. Mol Biol Cell. 25:2116. PubMed Ocklenburg T, et al. 2021. Sci Rep. 7199:11. PubMed Uriostegui–Arcos M, et al. 2020. Open Biol. 10:200050. PubMed Blake D, et al. 2022. Elife. 11:. PubMed Bujarrabal-Dueso A, et al. 2023. Nat Struct Mol Biol. 30:475. PubMed He W 2011. J Immunol. 186:5313. PubMed Angiari S, et al. 2020. Cell Metab. 31:391. PubMed Lindqvist B, et al. 2022. PLoS Pathog. 18:e1010555. PubMed Mascarenhas M, et al. 2016. Blood. 127: 2298 - 2309. PubMed Liang F, et al. 2021. Front Immunol. 12:658715. PubMed Jenke A, et al. 2013. PLoS One. 8:e55636. PubMed Petrillo C, et al. 2018. Cell Stem Cell. 1.527777778. PubMed Yin W, et al. 2022. Biomolecules. 12:. PubMed Khan E, et al. 2016. Sci Rep. 6:38104. PubMed Mihaly S, et al. 2014. PLoS One. 9:94982. PubMed Martel C, et al. 2011. PLoS One. 6:e18812. PubMed Saint Fleur–Lominy S, et al. 2018. Cell Rep. 24:3045. PubMed Gurrion C, et al. 2017. J Cancer. 2.323611111. PubMed Cha SE, et al. 2021. Oncoimmunology. 10:1899469. PubMed Oh J, et al. 2013. J Exp Med. 210:1069. PubMed Burbulla L, et al. 2014. Cell Death Dis. 5:1180. PubMed Peiris T, et al. 2016. Development. 143: 1697 - 1709.. PubMed Ramstead AG, et al. 2020. Cell Rep. 30:2889. PubMed Hewitt KJ et al. 2017. Developmental cell. 42(3):213-225 . PubMed Yabas M, et al. 2016. PLoS One. 11: 0146774. PubMed Lao Z, et al. 2015. PLoS One. 10: 0133895. PubMed Naegele M, et al. 2012. J Neuroimmunol. 242:60. PubMed Hejna M, et al. 2017. Sci Rep. 10.1038/s41598-017-12165-1. PubMed Gordy C, et al. 2011. Blood. 117:618. PubMed Khuat LT, et al. 2020. Sci Transl Med. 12:. PubMed Hasan S, et al. 2013. Blood. 122:1464. PubMed Wu Z, et al. 2019. Nat Metab. 1:1209. PubMed Chen X, et al. 2017. Autophagy. 1.204861111. PubMed Silginer M, et al. 2017. Cell Death Dis. 10.1038/cddis.2017.171. PubMed Kiss M, et al. 2020. Cancer Immunol Res. 9:309. PubMed Zhao B, et al. 2013. PLoS One. 8:77708. PubMed Guentsch A, et al. 2017. Mol Cell Biol. 37: e00236-16. PubMed Medina CB, et al. 2021. Immunity. 54(8):1715-1727.e7. PubMed Kranz P, et al. 2017. Cell Death Dis. 8:e2986. PubMed Bjørnstad R, et al. 2019. Mol Cancer Ther. 1.14375. PubMed Terlecki–Zaniewicz L, et al. 2018. Aging (Albany NY). 1.182638889. PubMed Wang X, et al. 2022. Elife. 11:. PubMed Sun Z, et al. 2022. Cancer Res. . PubMed Anderson CJ, et al. 2021. Nature. 596:262. PubMed León TE, et al. 2020. Cancer Discov. 10:998. PubMed Yabas M, et al. 2014. J Biol Chem. 289:19531. PubMed Mei Y, et al. 2020. Nat Commun. 2.661111111. PubMed Fontanals–Cirera B, et al. 2017. Mol Cell. 68:731. PubMed
RRID: AB_1279046 (BioLegend Cat. No. 640917) AB_1279044 (BioLegend Cat. No. 640918)
Biology Area: 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.