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48127
IL2-Rα/CD25 (PC61.5) Rat mAb (violetFluor™ 450 Conjugate)
Antibody Conjugates

IL2-Rα/CD25 (PC61.5) Rat mAb (violetFluor™ 450 Conjugate) #48127

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Flow cytometric analysis of live mouse splenocytes using IL2-Rα/CD25 (PC61.5) Rat mAb (violetFluor™ 450 Conjugate) and co-stained with CD4 (RM4-5) Rat mAb (APC Conjugate) #82116 (right), compared to concentration-matched Rat Isotype Control (violetFluor™ 450 Conjugate) (left).

To Purchase # 48127S
Product # Size Price
48127S
100 µg $ 189

Supporting Data

REACTIVITY M
SENSITIVITY
MW (kDa)
Isotype Rat IgG2b, kappa

Product Description

This Cell Signaling Technology antibody is conjugated to violetFluor™ 450 and tested in-house for direct flow cytometric analysis in mouse cells.

Product Usage Information

For optimal flow cytometry results, we recommend 0.5 μg of antibody per test.

Application Dilutions
Flow Cytometry 1:40

Storage:

Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2. This product is stable for 6 months when stored at 4ºC. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Live Cell Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
  2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted June 2017

revised August 2019

Protocol Id: 1504

Specificity / Sensitivity

IL2-Rα/CD25 (PC61.5) Rat mAb (violetFluor™ 450 Conjugate) recognizes endogenous levels of total IL2-Rα/CD25 protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Mouse

Source / Purification

This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography. The purified antibody was conjugated under optimal conditions, with unreacted dye removed from the preparation.

Background

Interleukin-2 (IL-2) is a T cell stimulatory cytokine best known for inducing T cell proliferation and NK cell proliferation and activation (1,2). IL-2 also promotes peripheral development of regulatory T cells (Tregs) (3,4). Conversely, IL-2 is involved in the activation-induced cell death (AICD) that is observed post T cell expansion by increasing levels of Fas on CD4+ T cells (5). The effects of IL-2 are mediated through a trimeric receptor complex consisting of IL-2Rα, IL-2Rβ, and the common gamma chain, γc (1,2). IL-2Rα binds exclusively to IL-2 with low affinity and increases the binding affinity of the whole receptor complex including IL-2Rβ and γc subunits. IL-15 also binds to IL-2Rβ (1,2). γc is used by other cytokines including IL-4, IL-7, IL-9, IL-15, and IL-21 (1,2). Binding of IL-2 initiates signaling cascades involving Jak1, Jak3, Stat5, and the PI3K/Akt pathways (1,2).

  1. Ma, A. et al. (2006) Annu Rev Immunol 24, 657-79.
  2. Gaffen, S.L. and Liu, K.D. (2004) Cytokine 28, 109-23.
  3. Fehérvari, Z. et al. (2006) Trends Immunol 27, 109-11.
  4. Antony, P.A. et al. (2006) J Immunol 176, 5255-66.
  5. Jaleco, S. et al. (2003) J Immunol 171, 61-8.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
violetFluor is a registered trademark of Tonbo Biosciences.