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Alexa Fluor® 647 anti-human CD8a Antibody

Pricing & Availability
Clone
C8/144B (See other available formats)
Regulatory Status
RUO
Other Names
T8, Leu2
Isotype
Mouse IgG1, κ
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Product Citations
publications
C8slash144B_A647_CD8a_Antibody_1_082918
Human paraffin-embedded tonsil tissue slices were prepared with a standard protocol of deparaffinization and rehydration. Antigen retrieval was done with Tris-Buffered Saline 1X (1.0M, pH7.4) at 95°C for 40 minutes. Tissue was washed with PBS/ 0.05% Tween 20 twice for five minutes, permeabilized with 0.5% Triton X-100 for ten minutes and blocked with 5% FBS and 0.2% gelatin for 30 minutes. Then, the tissue was stained with 5 µg/mL of Alexa Fluor® 647 anti-human CD8a (clone C8/144B) antibody (green) and Alexa Fluor® 594 anti-mouse/human Bcl-6 (clone IG191E/A8) antibody (red) at 4°C overnight. The image was captured with a 10X objective.
  • C8slash144B_A647_CD8a_Antibody_1_082918
    Human paraffin-embedded tonsil tissue slices were prepared with a standard protocol of deparaffinization and rehydration. Antigen retrieval was done with Tris-Buffered Saline 1X (1.0M, pH7.4) at 95°C for 40 minutes. Tissue was washed with PBS/ 0.05% Tween 20 twice for five minutes, permeabilized with 0.5% Triton X-100 for ten minutes and blocked with 5% FBS and 0.2% gelatin for 30 minutes. Then, the tissue was stained with 5 µg/mL of Alexa Fluor® 647 anti-human CD8a (clone C8/144B) antibody (green) and Alexa Fluor® 594 anti-mouse/human Bcl-6 (clone IG191E/A8) antibody (red) at 4°C overnight. The image was captured with a 10X objective.
  • C8slash144B_A647_CD8a_Antibody_2_082918
    Human peripheral blood mononuclear cells were surface stained with CD3 Brilliant Violet 421™ and CD4 PE, fixed with Fixation Buffer (Cat No. 420801), permeabilized with Intracellular Staining Permeabilization Wash Buffer (Cat. No. 421002) then intracellular stained with anti-human CD8a (clone C8/144B) Alexa Fluor® 647 (left) or mouse IgG1, κ Alexa Fluor® 647 isotype control (right). Cells were gated on the CD3+ population.
  • C8slash144B_A647_CD8a_Antibody_3_092121.png
    Paraformaldehyde-fixed (4%), 500 μm-thick mouse spleen section was processed according to the Ce3DTM Tissue Clearing Kit protocol (cat. no. 427701). The section was costained with anti-mouse CD21/CD35 (CR2/CR1) Antibody (clone 7E9) Alexa Fluor® 594 at 5 µg/mL (green), and anti-mouse CD8a Antibody (clone C8/144B) Alexa Fluor® 647 at 5 µg/mL (magenta) and counterstained with DAPI (blue). The section was then optically cleared and mounted in a sample chamber. The image was captured with a 10X objective using Zeiss 780 confocal microscope and processed by Imaris image analysis software.
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372906 100 µg 198€
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Description

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 α3 domain of MHC class I and the cytoplasmic CXCP motif binds the tyrosine kinase p56 Lck.

Product Details
Technical data sheet

Product Details

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.

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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)
  1. McQuitty E, et al. 2014. J. Cutan. Pathol. 41:88. (IHC-P)
  2. Petukhova L, et al. 2010. Nature 466:113. (IHC-F)
  3. Clement M, et al. 2011. J. Immunol. 187:654. (Activ)
  4. MarTchal R, et al. 2010. BMC Cancer 10:340. (FC)
  5. Pontiggia L, et al. 2008. J. Invest. Dermatol. 129:480. (WB)
Product Citations
  1. Wang S, et al. 2022. FASEB J. 36:e22336. PubMed
  2. Korsunsky I, et al. 2022. Med. 3:481. PubMed
  3. Pelka K, et al. 2021. Cell. 184:4734. PubMed
  4. Chen B, et al. 2021. Cell. 184:6262. PubMed
RRID
AB_2650712 (BioLegend Cat. No. 372906)

Antigen Details

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 View all products for this Gene ID
UniProt
View information about CD8a on UniProt.org

Related FAQs

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

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.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

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.

Are other fluorophores compatible with IBEX?

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.

The same antibody works in one tissue type but not another. What is happening?

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.

How can I be sure the staining I’m seeing in my tissue is real?

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.

Go To Top Version: 3    Revision Date: 09.21.2021

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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