Biolegend, 100738, Brilliant Violet 421™ anti-mouse CD8a Antibody, 500microl

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.
500microl
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 and BSA (origin USA).
Preparation: The antibody was purified by affinity chromatography and conjugated with Brilliant Violet 421™ under optimal conditions.
Concentration: µg sizes: 0.2 mg/mLµL sizes: lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
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 - 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 immunofluorescent staining using the µg size, the suggested use of this reagent is ≤0.5 µg per million cells in 100 µl volume. For immunofluorescent staining using µl sizes, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood. It is recommended that the reagent be titrated for optimal performance for each application. Brilliant Violet 421™ excites at 405 nm and emits at 421 nm. The standard bandpass filter 450/50 nm is recommended for detection. Brilliant Violet 421™ is a trademark of Sirigen Group Ltd. Learn more about Brilliant Violet™. This product is subject to proprietary rights of Sirigen Inc. and is made and sold under license from Sirigen Inc. The purchase of this product conveys to the buyer a non-transferable right to use the purchased product for research purposes only. This product may not be resold or incorporated in any manner into another product for resale. Any use for therapeutics or diagnostics is strictly prohibited. This product is covered by U.S. Patent(s), pending patent applications and foreign equivalents.
Excitation Laser: Violet Laser (405 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: 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).
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RRID: AB_10897101 (BioLegend Cat. No. 100737) AB_2562558 (BioLegend Cat. No. 100753) AB_11204079 (BioLegend Cat. No. 100738)
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: What is the F/P ratio range of our BV421™ format antibody reagents?
A: It is lot-specific. On average it ranges between 2-4.
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.