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

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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)
Isotype Hamster (Armenian) IgG

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 Dilutions
Flow Cytometry 1:200

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

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.

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.