Invitrogen™ CD25 Monoclonal Antibody (P4A10), Super Bright™ 600, eBioscience™

Catalog No.	Quantity
63-025-042	100 Tests

Catalog No. 63-025-042 Supplier Invitrogen™ Supplier No. 63025042

Description

Mouse Monoclonal Antibody

Description: This P4A10 monoclonal antibody reacts with canine CD25, the 55-kDa interleukin-2 receptor alpha (IL-2Ralpha) chain. As in humans and mice, CD25 in dogs is expressed by early T and B lineage progenitors as well as by activated mature T and B lymphocytes. CD25 associates with the IL-2R beta (CD122) and gamma (CD132) chains to form the high-affinity IL-2 receptor. On its own, CD25 binds IL-2 with low affinity. Along with CD4 and Foxp3, CD25 is a major marker used to define regulatory T cells. The P4A10 antibody has been reported to recognize the same epitope as the anti-human CD25 monoclonal antibody ACT-1, but with greater affinity. Applications Reported: This P4A10 antibody has been reported for use in flow cytometric analysis. Applications Tested: This P4A10 antibody has been pre-titrated and tested by flow cytometric analysis of normal canine peripheral blood cells. This can be used at 5 μL (0.125 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. Super Bright 600 is a tandem dye that can be excited with the violet laser line (405 nm) and emits at 600 nm. We recommend using a 610/20 bandpass filter. Please make sure that your instrument is capable of detecting this fluorochrome. When using two or more Super Bright dye-conjugated antibodies in a staining panel, it is recommended to use Super...

CD25 (IL2 receptor alpha chain/IL2RA) is a cytokine that plays a role in the proliferation of T and B lymphocytes. The receptor of this cytokine (IL2RA) is a heterotrimeric protein complex with a gamma chain also shared by interleukin 4 (IL4) and interleukin 7 (IL7). IL2RA, IL2R beta chain (IL2RB), and the IL2R gamma chain (IL2RG), constitute the high-affinity IL2 receptor. Homodimeric IL2RA chains result in low-affinity receptor, while homodimeric IL2RB chains produce a medium-affinity receptor. The expression of IL2 in mature thymocytes is monoallelic, which represents an unusual regulatory mode for controlling the precise expression of a single gene. IL2 is primarily produced by mature T cells. IL2 plays an important role as a growth factor, differentiation factor, and regulator of cell death. IL-2 stimulates the proliferation of B cells, augments natural killer cell activity, and inhibits granulocyte macrophage colony formation. The targeted disruption of a similar gene in mice leads to ulcerative colitis-like disease, which suggests a role in the immune response to antigenic stimuli. Mutations in this gene are associated with interleukin 2 receptor alpha deficiency.

Specifications

• Antigen: CD25
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: P4A10
• Concentration: 5 μL/Test
• Conjugate: Super Bright 600
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: IL2RA
• Gene Accession No.: O62802
• Gene Alias: ADA; ADA1; Adenosine aminohydrolase; adenosine deaminase; CD25; CD25 antigen; cytokine receptor; IDDM10; IL 2 RA; IL 2 receptor; IL 2R; IL 2R subunit alpha; IL2 RA; IL2 RA antibody; IL2 receptor; il-2 receptor alpha; IL-2 Receptor alpha chain; IL-2 receptor alpha subunit; IL2 receptor subunit alpha; IL-2 receptor subunit alpha; IL2R; IL-2R alpha; IL-2R alpha chain; IL2R subunit alpha; IL-2R subunit alpha; Il2ra; IL-2RA; IL-2-RA; IL2-RA; Il2ra antibody; IL2RAC; IMD41; interleukin 2 receptor; interleukin 2 receptor alpha chain; interleukin 2 receptor subunit alpha; interleukin 2 receptor, alpha; interleukin 2 receptor, alpha chain; interleukin-2 receptor alpha; interleukin-2 receptor alpha chain; interleukin-2 receptor alpha subunit; interleukin-2 receptor beta chain; Interleukin2 receptor subunit alpha; interleukin-2 receptor subunit alpha; Ly-43; p55; p55 chain; sIL 2R; soluble IL 2 receptor; TAC antigen; TCGFR
• Gene Symbols: IL2RA
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 403870
• Target Species: Canine
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG1 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.