Biolegend, 100727, Alexa Fluor® 647 anti-mouse CD8a Antibody, 25μg

CD8, also known as Lyt-2, Ly-2, or T8, consists of disulfide-linked α and β chains that form the α(CD8a)/β(CD8b) heterodimer and α/α homodimer. CD8a is a 34 kD protein that belongs to the immunoglobulin family. The CD8 α/β heterodimer is expressed on the surface of most thymocytes and a subset of mature TCR α/β T cells. CD8 expression on mature T cells is non-overlapping with CD4. The CD8 α/α homodimer is expressed on a subset of γ/δ TCR-bearing T cells, NK cells, intestinal intraepithelial lymphocytes, and lymphoid dendritic cells. CD8 is an antigen co-receptor on T cells that interacts with MHC class I on antigen-presenting cells or epithelial cells. CD8 promotes T cell activation through its association with the TCR complex and protein tyrosine kinase lck.
25μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: Mouse thymus or spleen
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration: 0.5 mg/mL
Storage & Handling: The antibody 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 IHC-F, 3D IHC - Verified SB - Community verified SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤ 0.25 µg per million cells in 100 µL volume. For immunohistochemistry on frozen tissue sections, a concentration range of 2.5 - 5.0 μg/mL is suggested. For 3D immunohistochemistry on formalin-fixed tissues, a concentration of 5.0 µg/mL is suggested. It is recommended that the reagent be titrated for optimal performance for each application. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633nm / 635nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Red Laser (633 nm)
Application Notes: Clone 53-6.7 antibody competes with clone 5H10-1 antibody for binding to thymocytes3. The 53-6.7 antibody has been reported to block antigen presentation via MHC class I and inhibit T cell responses to IL-2. This antibody has also been used for depletion of CD8a+ cells. Additional reported applications (for the relevant formats) include: immunoprecipitation1,3, in vivo and in vitro cell depletion2,10,15, inhibition of CD8 T cell proliferation3, blocking of cytotoxicity3,4, immunohistochemical staining5,6 of acetone-fixed frozen sections and zinc-fixed paraffin-embedded sections, and spatial biology (IBEX)29,30. Clone 53-6.7 is not recommended for immunohistochemistry of formalin-fixed paraffin sections. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays or in vivo studies (Cat No. 100746).
Additional Product Notes: For use in spatial biology, this antibody has been demonstrated for use in immunohistochemistry using IBEX (Reported in the literature, not verified in house) and the NanoString GeoMx® Digital Spatial Profiler. IBEX: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH). NanoString GeoMx®: This product has been verified for IHC-F (Immunohistochemistry - frozen tissue sections) on the NanoString GeoMx® Digital Spatial Profiler. The GeoMx® enables researchers to perform spatial analysis of protein and RNA targets in FFPE and fresh frozen human and mouse samples. For more information about our spatial biology products and the GeoMx® platform, please visit our spatial biology page.
Application References(PubMed link indicates BioLegend citation): Ledbetter JA, et al. 1979. Immunol. Rev. 47:63. (IHC, IP) Hathcock KS. 1991. Current Protocols in Immunology. 3.4.1. (Deplete) Takahashi K, et al. 1992. P. Natl. Acad. Sci. USA 89:5557. (Block, IP) Ledbetter JA, et al. 1981. J. Exp. Med. 153:1503. (Block) Hata H, et al. 2004. J. Clin. Invest. 114:582. (IHC) Fan WY, et al. 2001. Exp. Biol. Med. 226:1045. (IHC) Shih FF, et al. 2006. J. Immunol. 176:3438. (FC) Kamimura D, et al. 2006. J. Immunol. 177:306. Bouwer HGA, et al. 2006. P. Natl. Acad. Sci. USA 103:5102. (FC, Deplete) Kao C, et al. 2005. Int. Immunol. 17:1607. PubMed Ko SY, et al. 2005. J. Immunol. 175:3309. (FC) PubMed Rasmussen JW, et al. 2006. Infect. Immun. 74:6590. PubMed Lee CH, et al. 2009. Clin. Cancer Res. PubMed Geiben-Lynn R, et al. 2008. Blood 112:4585. (Deplete) PubMed Kingeter LM, et al. 2008. J. Immunol. 181:6244. PubMed Guo Y, et al. 2008. Blood 112:480. PubMed Andrews DM, et al. 2008. J. Virol. 82:4931. PubMed Britschqui MR, et al. 2008. J. Immunol. 181:7681. PubMed Kenna TJ, et al. 2008. Blood 111:2091. PubMed Jordan JM, et al. 2008. Infect. Immun. 76:3717. PubMed Todd DJ, et al. 2009. J. Exp. Med. 206:2151. PubMed Bankoti J, et al. 2010. Toxicol. Sci. 115:422. (FC) PubMed Medyouf H, et al. 2010. Blood 115:1175. PubMed Riedl P, et al. 2009. J. Immunol. 183:370. PubMed Apte SH, et al. 2010. J. Immunol. 185:998. PubMed Bankoti J, et al. 2010. Toxicol. Sci. 115:422. (FC) PubMed del Rio ML, et al. 2011. Transpl. Int. 24:501. (FC) PubMed Cui L, et al. 2015. J Control Release. 206:220. PubMed Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Djoki&cacute; V, et al. 2019. Front Immunol. 9:2891. PubMed Yu X, et al. 2020. Nat Commun. 11:1110. PubMed Minute L, et al. 2020. J Immunother Cancer. 8:. PubMed Briukhovetska D, et al. 2023. Immunity. 56:143. PubMed Rodriguez-Ruiz ME, et al. 2023. J Immunother Cancer. 11: . PubMed Scherer S, et al. 2023. Nat Immunol. 24:501. PubMed Feist M, et al. 2021. Cancer Gene Ther. 28:98. PubMed Allen SD, et al. 2021. Biomaterials. 269:120635. PubMed Parlane N, et al. 2013. Vet Immunol Immunopathol. 30:122. PubMed Hu HJ, et al. 2020. Cell Death Dis. 1.168055556. PubMed Baptista AP et al. 2019. Immunity. 50(5):1188-1201 . PubMed Kim K, et al. 2009. PLoS One. 4:e7738. PubMed Park JJ, et al. 2021. Nat Commun. 12:1222. PubMed Tang B, et al. 2020. Clin Cancer Res. 26:2216. PubMed Peng Z, et al. 2021. STAR Protocols. 2(2):100595. PubMed Najjar YG, et al. 2019. JCI Insight. 4:. PubMed Etxeberria I, et al. 2020. Cancer Cell. 36(6):613-629. PubMed Dai B, et al. 2022. Theranostics. 12:7603. PubMed Ma YV, et al. 2021. MAbs. 13:2003281. PubMed Schuhmann MK, et al. 2021. J Neuroinflammation. 18:46. PubMed Seedhom M, et al. 2016. J Immunol. 197: 1498 - 1506. PubMed Young R, et al. 2015. Proc Natl Acad Sci U S A. 112: 13447 - 13454. PubMed Qi S et al. 2016. eLife. 5 pii: e14756. PubMed Kim SH, et al. 2021. Cell Reports. 35(2):108995. PubMed Wang F, et al. 2019. MAbs. 12:1685350. PubMed Liang J, et al. 2020. Sci Adv. 6:eabc3646. PubMed Britschgi M, et al. 2008. J Immunol. 181:7681. PubMed Koikawa K, et al. 2021. Cell. 184:4753. PubMed Yu X, et al. 2019. Nat Commun. 10:574. PubMed Matryba P, et al. 2020. J Immunol. 1395:204. PubMed Yu M, et al. 2021. Molecular Cell. 81(6):1216-1230.e9. PubMed Tanaka Y, et al. 2017. J Immunol. 199:4016. PubMed von Roemeling CA, et al. 2020. Nat Commun. 11:1508. PubMed Zhang Y, et al. 2021. Commun Biol. 344:4. PubMed Werner A, et al. 2021. iScience. 24:103076. PubMed Rui J, et al. 2021. Nat Commun. 12:5074. PubMed Chryplewicz A, et al. 2022. Cancer Cell. 40:1111. PubMed Yin Y, et al. 2016. J Biol Chem. 291: 6923 - 6935. PubMed Zhou Y, et al. 2016. Sci Rep. 6:28140. PubMed Xiao M, et al. 2022. Mol Oncol. 16:1026. PubMed Richardson M, et al. 2014. PLoS Negl Trop Dis. 8:2825. PubMed Rasmussen J, et al. 2006. Infect Immun. 74:6590. PubMed
RRID: AB_493424 (BioLegend Cat. No. 100727) AB_389326 (BioLegend Cat. No. 100724)
Structure: Ig superfamily, CD8α chain, 34 kD
Distribution: Most thymocytes, T cell subset, some NK cells, lymphoid dendritic cells
Function: Co-receptor for TCR
Ligand/Receptor: MHC class I molecule
Antigen References: 1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press. 2. Zamoyska R. 1994. Immunity 1:243. 3. Ellmeier W, et al. 1999. Annu. Rev. Immunol. 17:523.
Gene ID: 12525
UniProt: View information about CD8alpha on UniProt.org
Clone: 53-6.7
Regulatory Status: RUO
Other Names: T8, Lyt2, Ly-2
Isotype: Rat IgG2a, κ
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.