Invitrogen™ CD8a Monoclonal Antibody (53-6.7), Super Bright™ 436, eBioscience™, Invitrogen™

Catalog No.	Quantity
62-008-182	100 μg
62-008-180	25 μg

Catalog No. 62-008-182 Supplier Invitrogen™ Supplier No. 62008182

Description

Rat Monoclonal Antibody

Description: The 53-6.7 monoclonal antibody reacts with the mouse CD8a molecule. CD8a is an approximately 32-34 kDa cell surface receptor expressed either as a heterodimer with the CD8 beta chain (CD8 alpha beta) or as a homodimer (CD8 alpha alpha). A majority of thymocytes and a subpopulation of mature alpha beta TCR T cells express CD8 alpha beta while gamma delta TCR T cells, a subpopulation of intestinal intraepithelial lymphocytes (IELs) and dendritic cells express CD8 alpha alpha. CD8 binds to MHC class I and through its association with protein tyrosine kinase p56lck plays a role in T cell development and activation of mature T cells. Applications Reported: This 53-6.7 antibody has been reported for use in flow cytometric analysis. Applications Tested: This 53-6.7 antibody has been tested by flow cytometric analysis of mouse splenocytes. This may be used at less than or equal to 0.25 μg per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Super Bright 436 can be excited with the violet laser line (405 nm) and emits at 436 nm. We recommend using a 450/50 bandpass filter, or equivalent. Please make sure that your instrument is capable of detecting this fluorochrome.

CD8, also known as cluster of differentiation 8, is a type I transmembrane glycoprotein of the immunoglobulin family that plays a crucial role in T cell differentiation, activation, and signal transduction. It is expressed as either a heterodimer (CD8 alpha beta) or a homodimer (CD8 alpha alpha). The CD8 alpha beta form is predominantly found on the majority of thymocytes and a subpopulation of mature alpha beta TCR T cells, while the CD8 alpha alpha form is expressed on gamma delta TCR T cells, a subset of intestinal intraepithelial lymphocytes (IELs), and dendritic cells. CD8 functions as a co-receptor for major histocompatibility complex class I (MHC-I) molecules, working alongside the T cell receptor (TCR). The CD8 alpha chain is essential for binding to MHC-I. CD8 is also expressed on a subset of T cells, NK cells, monocytes, and dendritic cells as disulfide-linked homodimers of CD8 alpha. Upon ligation of MHC-I/peptide complexes presented by antigen-presenting cells (APCs), CD8 recruits lymphocyte-specific protein tyrosine kinase (Lck), leading to lymphokine production, increased motility, and activation of cytotoxic T lymphocytes (CTLs). Activated CTLs are vital for clearing pathogens and tumor cells. The differentiation of naive CD8+ T cells into CTLs is strongly enhanced by cytokines such as IL-2, IL-12, and TGF-beta1. Through its interactions with MHC-I and association with protein tyrosine kinase p56lck, CD8 plays a significant role in T cell development and the activation of mature T cells.

Specifications

• Antigen: CD8a
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: 53-6.7
• Concentration: 0.2 mg/mL
• Conjugate: Super Bright 436
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: Cd8a
• Gene Accession No.: P01731, P10300
• Gene Alias: BB154331; CD8; CD8 alpha; CD8 alpha chain; CD8 alpha chain precursor; CD8 antigen; CD8 antigen 32 kDa chain; CD8 antigen 37 kDa chain; CD8 antigen alpha polypeptide; CD8 antigen alpha protein; CD8 antigen alpha protein precursor; CD8 antigen alpha-chain; CD8 antigen beta polypeptide; CD8 antigen beta polypeptide precursor; CD8 antigen beta-chain; CD8 antigen, alpha chain; CD8 antigen, alpha polypeptide; CD8 antigen, alpha polypeptide (p32); CD8 antigen, alpha-chain; CD8 antigen, beta chain; CD8 antigen, beta chain 1; CD8 antigen, beta polypeptide; CD8 antigen, beta polypeptide 1 (p37); CD8 antigen, beta-chain; CD8 beta; CD8 beta chain; CD8 beta-2; CD8a; CD8A antigen alpha; CD8a molecule; CD8A; T-cell surface glycoprotein; CD8alpha; CD8B; CD8b antigen; CD8b molecule; CD8b molecule pseudogene; Cd8b1; CD8beta; CD8BP; fCD8; LEU2; Leu-2; Leu2 T-lymphocyte antigen; leu-2a; Ly-2; LY3; Ly-3; Ly-35; Ly-B; Ly-C; Lymphocyte antigen 3; Lyt2; Lyt-2; Lyt-2.1 lymphocyte differentiation antigen (AA at 100); Lyt3; Lyt-3; MAL; membrane glycoprotein; membrane protein; OKT8 T-cell antigen; OX-8 membrane antigen; p32; P37; RHACD8-4; T cell co-receptor; T lymphocyte surface glycoprotein beta chain; T8 T-cell antigen; T-cell antigen Leu2; T-cell membrane glycoprotein Ly-3; T-cell surface glycoprotein; T-cell surface glycoprotein CD8 alpha chain; T-cell surface glycoprotein CD8 beta chain; T-cell surface glycoprotein Lyt-2; T-cell surface glycoprotein Lyt-3; T-cell surface molecule; T-lymphocyte differentiation antigen T8/Leu-2; type I transmembrane glycoprotein
• Gene Symbols: Cd8a, Cd8b1
• Host Species: Rat
• Purification Method: Affinity chromatography
• Quantity: 100 μg
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 12525, 12526
• Target Species: Mouse
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2a κ

Frequently Asked Questions (FAQs)

Can I use the OneComp and UltraComp eBeads microspheres with Super Bright-conjugated antibodies?

UltraComp eBeads microspheres (Cat. No. 01-2222) are recommended for use with Super Bright dyes.
Note: Super Bright Staining Buffer (Cat. No. SB-4400) is not compatible with UltraComp eBeads microspheres (Cat. No. 01-2222-41, 00-2222-42). If using UltraComp eBeads microspheres as a compensation tool, solely use Flow Cytometry Stain Buffer (Cat. No. 00-4222-26, 00-4222-57) for any antibody dilutions.

In some experiments, we have observed that compensation values for Super Bright 780- and Brilliant Violet 785- or Brilliant Violet 786-conjugated antibodies are higher in the violet 450/50 channel when using UltraComp eBeads microspheres as compared to single-color stained cells. In such circumstances, we would recommend setting compensation with cells. We have also observed this in some experiments using AbC Total Antibody Compensation beads, both with Super Bright 780 and Brilliant Violet 786. We have not tested Brilliant Violet 785 with the AbC beads.

Can I prepare an antibody cocktail containing Super Bright Staining Buffer and Super Bright-conjugated antibodies ahead of time?

We recommend that the antibody cocktails containing Super Bright-conjugated antibodies and Super Bright Staining Buffer are prepared fresh prior to staining. Discard any unused portions. We do not recommend overnight storage of prepared cocktails.

Can I fix my cells after staining with Super Bright-conjugated antibodies? How long can fixed cells be stored prior to analysis?

Samples that have been stained with antibodies conjugated to Super Bright dyes may be stored for up to three days, at 2-8°C, in the dark, using either IC Fixation Buffer (Cat. No. 00-8222) or 1-step Fix/Lyse Buffer (Cat. No. 00-5333) with no significant effect on brightness or compensation.

Are the Super Bright dyes sensitive to methanol fixation?

Super Bright dyes are stable in methanol-based fixation buffers.

Can Super Bright-conjugated antibodies be used in combination with intracellular (IC Fixation/Permeabilization) or intranuclear (Foxp3 fixation/permeabilization) staining?

Yes, Super Bright-conjugated antibodies are stable in formaldehyde-based fixation buffers and permeabilization buffers, such as the IC Fixation and Permeabilization Buffer Set (Cat. No. 88-8824) and the Foxp3/Transcription Factor Staining Buffer Set (Cat. No. 00-5523).

Which laser do I use to detect Super Bright-conjugated antibodies?

The violet laser (405 nm) should be used to excite Super Bright-conjugated antibodies.

Can the Super Bright Staining Buffer be used with other polymer dyes?

Yes, the Super Bright Staining Buffer (Cat. No. SB-4400) is compatible with other polymer dyes (i.e., Brilliant Violet dyes) and is useful for minimizing any non-specific polymer interactions when two or more of these dyes are used in combination.

Can Super Bright-conjugated antibodies be used in combination with other polymer dyes? What buffer should I use when using more than one Super Bright or polymer dye?

When using two or more Super Bright dyes, or when using Super Bright dyes in combination with other polymer dyes (i.e., Brilliant Violet dyes), we recommend using the Super Bright Staining Buffer (Cat. No. SB-4400) to minimize any non-specific polymer interactions between these fluorochromes. For specific instructions for use, please refer to the product technical data sheet.

Are there recommended tubes for staining with Super Bright-conjugated antibodies?

We recommend using polystyrene tubes (for example, FACS tubes) for staining with your Super Bright-conjugated antibodies. If using polypropylene (for example, Eppendorf tubes), protecting from light is critical.

Is there a specific buffer I should use when staining with Super Bright-conjugated antibodies?

No special buffer is required when using a single Super Bright-conjugated antibody in a panel.
When using more than one Super Bright dye, or when using Super Bright dyes in combination with other polymer dyes (i.e., Brilliant Violet dyes), we recommend using the Super Bright Staining Buffer (Cat. No. SB-4400) to minimize any non-specific polymer interactions between these fluorochromes. For specific instructions for use, please refer to the product technical data sheet.

How does the protocol for staining with Super Bright-conjugated antibodies differ from staining with conventional organic fluorochrome and eVolve conjugated antibodies?

Antibodies conjugated to Super Bright dyes can be used similarly to traditional fluorochromes. If multiple Super Bright dyes are used in combination with each other or in combination with other polymer dyes (i.e., Brilliant Violet dyes), then the use of Super Bright Staining Buffer (Cat. No. SB-4400) is recommended to minimize any non-specific polymer interactions between these fluorochromes. For specific instructions for use, please refer to the product technical data sheet.

Can the Super Bright dyes be used for non-flow applications?

Super Bright dyes have not been tested for applications other than flow cytometry.

What is the difference between the Super Bright and eVolve dyes?

Super Bright dyes are fluorochromes based on polymer technology and are excited by the violet laser. In contrast, eVolve dyes are based on Qdot nanocrystal technology and are maximally excited by the UV laser, although they may also be excited by violet, blue, yellow-green, and, depending on the eVolve, the red laser lines. The general shape of the emission spectra for Super Bright dyes will be more similar to traditional fluorochromes, while eVolve dyes have extremely narrow emission spectra. Please contact Tech Support (techsupport@thermofisher.com) for more information.

What are the Super Bright dyes?

eBioscience Super Bright dyes are a series of patent-pending fluorochromes that are based upon a fluorescent polymer and its tandems. Super Bright dyes have been developed for use in flow cytometry and can be excited by the violet laser (405 nm). The peak emission of each Super Bright dye is indicated by the number in the product description, e.g., "Super Bright 600" has a peak emission of 600 nm.

Are the Super Bright Dyes photo-labile?

As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.