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39443
CD69 (H1.2F3) Hamster mAb (FITC Conjugate)
Antibody Conjugates
Monoclonal Antibody
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CD69 (H1.2F3) Hamster mAb (FITC Conjugate) #39443

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Flow Cytometry Image 1: CD69 (H1.2F3) Hamster mAb (FITC Conjugate)
Flow cytometric analysis of live mouse splenocytes, untreated (blue) or treated with Concanavalin A (3 µg/ml, overnight; green), using CD69 (H1.2F3) Hamster mAb (FITC Conjugate) (solid lines) or concentration-matched Armenian Hamster Isotype Control (FITC Conjugate) (dashed lines).
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To Purchase # 39443S
Product # Size Price
39443S		 100 µg $ 99

Custom Formulation

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Hamster (Armenian) IgG

Application Key:

  • WB-Western Blot
  • 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
  • Vir-Virus
  • 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 mouse cells.

Product Usage Information

For optimal flow cytometry results, we recommend 0.25 μg of antibody per test.

Application Dilution
Flow Cytometry 1:200

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

CD69 (H1.2F3) Hamster mAb (FITC Conjugate) recognizes endogenous levels of total CD69 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

CD69, also known as Leu-23, is a type II transmembrane glycoprotein that is expressed on the surface of T cells, B cells, and NK cells (1,2). This phosphorylated disulfide-linked 28 to 32-kDa homodimer is constitutively expressed on a subset of thymocytes and platelets. It also acts as an activation antigen of lymphocytes, NK cells, neutrophils, and eosinophils (1-6). Studies have shown that stimulation of the T cell receptor (TCR) increases the expression of CD69 on the cell surface. The ability to detect the level of CD69 expression after TCR activation makes CD69 an ideal indicator of T cell activation (1).  The H1.2F3 antibody is widely used as a marker for T cell activation (7).
  1. Testi, R. et al. (1989) J Immunol 142, 1854-60.
  2. Marzio, R. et al. (1997) J Leukoc Biol 62, 349-55.
  3. Lanier, L.L. et al. (1988) J Exp Med 167, 1572-85.
  4. Testi, R. et al. (1988) J Immunol 141, 2557-63.
  5. Hartnell, A. et al. (1993) Immunology 80, 281-6.
  6. Gavioli, R. et al. (1992) Cell Immunol 142, 186-96.
  7. Sobel, E.S. et al. (1993) J Immunol 150, 673-82.

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.