Biolegend, 372906, Alexa Fluor® 647 anti-human CD8a Antibody, 100μg

CD8a is a 32-34 kD type I glycoprotein. It forms a homodimer (CD8a/a) or heterodimer (CD8a/b) with CD8b. CD8, also known as T8 and Leu2, is a member of the immunoglobulin superfamily found on the majority of thymocytes, a subset of peripheral blood T cells, and NK cells (which express almost exclusively CD8a homodimers). CD8 acts as a co-receptor with MHC class I-restricted T cell receptors in antigen recognition and T cell activation, and has been shown to play a role in thymic differentiation. Two domains in CD8a are important for function: the extracellular IgSF domain binds the α 3 domain of MHC class I and the cytoplasmic CXCP motif binds the tyrosine kinase p56 Lck.
100μg
Verified Reactivity: Human, Mouse, Rat
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: A 13 amino acid synthetic peptide from the C-terminal cytoplasmic domain of the alpha chain of the human CD8 molecule.
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: IHC-P - Quality tested ICFC, 3D IHC - Verified SB - Community verified
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 ≤ 5.0 - 10 µg per million cells in 100 µl volume. For flow cytometric staining, the suggested use of this reagent is ≤ 0.25 µg per million cells in 100 µl volume. 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 633 nm / 635 nm. 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: Additional reported applications (for the relevant formats) include activation3, flow cytometry4, immunohistochemical staining of frozen tissue sections2, and Western blotting5.
Additional Product Notes: This product has been verified for IHC-P (Immunohistochemistry - formalin-fixed paraffin-embedded tissues) 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): McQuitty E, et al. 2014. J. Cutan. Pathol. 41:88. (IHC-P) Petukhova L, et al. 2010. Nature 466:113. (IHC-F) Clement M, et al. 2011. J. Immunol. 187:654. (Activ) MarTchal R, et al. 2010. BMC Cancer 10:340. (FC) Pontiggia L, et al. 2008. J. Invest. Dermatol. 129:480. (WB)
Product Citations: Korsunsky I, et al. 2022. Med. 3:481. PubMed Wang S, et al. 2022. FASEB J. 36:e22336. PubMed Pelka K, et al. 2021. Cell. 184:4734. PubMed Chen B, et al. 2021. Cell. 184:6262. PubMed
RRID: AB_2650712 (BioLegend Cat. No. 372906)
Structure: Ig superfamily, homodimer or heterodimer with CD8β.
Distribution: Majority of thymocytes, T cell subset, NK cells.
Function: MHC class I co-receptor, thymic differentiation, T-cell activation.
Ligand/Receptor: MHC class I molecules.
Cell Type: NK cells, T cells, Thymocytes
Biology Area: Immunology
Molecular Family: CD Molecules
Antigen References: 1. McQuitty E, et al. 2014. J. Cutan. Pathol. 41:88.
Gene ID: 100387674
UniProt: View information about CD8a on UniProt.org
Clone: C8/144B
Regulatory Status: RUO
Other Names: T8, Leu2
Isotype: Mouse IgG1, 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.