Biolegend, 100211, Alexa Fluor® 647 anti-mouse CD3 Antibody, 25μg

CD3, also known as T3, is a member of the Ig superfamily and primarily expressed on T cells, NK-T cells, and at different levels on thymocytes during T cell differentiation. CD3 is composed of CD3ε, δ, γ and ζ chains. It forms a TCR complex by associating with TCR α/β or γ/δ chains. CD3 plays a critical role in TCR signal transduction, T cell activation, and antigen recognition by binding the peptide/MHC antigen complex
25μg
Verified Reactivity: Mouse
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: γδTCR-positive T-T hybridoma D1
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
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 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 application (for relevant formats) include: spatial biology (IBEX)1,2.
Additional Product Notes: 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): 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
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RRID: AB_389323 (BioLegend Cat. No. 100209)
Structure: Ig superfamily, CD3/TCR, 20 kD
Distribution: Thymocytes (differentiation dependent), mature T cells, NK-T cells
Function: Antigen recognition, TCR signal transduction, T cell activation
Ligand/Receptor: Peptide antigen/MHC-complex
Antigen References: 1. Barclay A, et al. 1997. The Leukocyte Antigen FactsBook Academic Press. 2. Davis MM. 1990. Annu. Rev. Biochem. 59:475. 3. Weiss A, et al. 1994. Cell 76:263.
Gene ID: 12502
UniProt: View information about CD3 on UniProt.org
Clone: 17A2
Regulatory Status: RUO
Other Names: T cell antigen receptor complex, T3
Isotype: Rat IgG2b, 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.