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23672
CD45RA (HI100) Mouse mAb (FITC Conjugate)
Antibody Conjugates
Monoclonal Antibody

CD45RA (HI100) Mouse mAb (FITC Conjugate) #23672

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Flow Cytometry Image 1: CD45RA (HI100) Mouse mAb (FITC Conjugate)

Flow cytometric analysis of live human peripheral blood mononuclear cells using CD45RA (HI100) Mouse mAb (FITC Conjugate) (solid line) compared to concentration-matched Mouse Isotype Control (FITC Conjugate) (dashed line).

To Purchase # 23672S
Product # Size Price
23672S
500 µl  (100 tests) $ 89

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG2b kappa

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to FITC and tested in-house for direct flow cytometric analysis in human cells.

Product Usage Information

Application Dilution
Flow Cytometry 1:20

Storage

Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2. 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 for a full 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 June 2020

Protocol Id: 1504

Specificity / Sensitivity

CD45RA (HI100) Mouse mAb (FITC Conjugate) recognizes endogenous levels of total CD45RA protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Human

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

The protein phosphatase (PTP) receptor CD45 is a type I transmembrane protein comprised of a pair of intracellular tyrosine phosphatase domains and a variable extracellular domain generated by alternative splicing (1). The catalytic activity of CD45 is a function of the first phosphatase domain (D1) while the second phosphatase domain (D2) may interact with and stabilize the first domain, or recruit/bind substrates (2,3). CD45 interacts directly with antigen receptor complex proteins or activates Src family kinases involved in the regulation of T- and B-cell antigen receptor signaling (1). Specifically, CD45 dephosphorylates Src-family kinases Lck and Fyn at their conserved negative regulatory carboxy-terminal tyrosine residues and upregulates kinase activity. Conversely, studies indicate that CD45 can also inhibit Lck and Fyn by dephosphorylating their positive regulatory autophosphorylation site. CD45 appears to be both a positive and a negative regulator that conducts signals depending on specific stimuli and cell type (1). Human leukocytes including lymphocytes, eosinophils, monocytes, basophils, and neutrophils express CD45, while erythrocytes and platelets are negative for CD45 expression (4).

Several isoforms of CD45 are generated through alternative splicing in a cell type-specific and activation state-specific manner. The HI100 antibody is widely used as a leukocyte marker for naïve and activated T cells. Naïve T cells are positive for CD45RA, where activated T cells are negative for CD45RA (5).

  1. Huntington, N.D. and Tarlinton, D.M. (2004) Immunol Lett 94, 167-74.
  2. Felberg, J. and Johnson, P. (2000) Biochem Biophys Res Commun 271, 292-8.
  3. Kashio, N. et al. (1998) J Biol Chem 273, 33856-63.
  4. Wang, Y. and Johnson, P. (2005) J Biol Chem 280, 14318-24.
  5. Cosenza-Nashat, M.A. et al. (2006) Brain Pathol 16, 256-65.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.