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19805
CD27 (O323) Mouse mAb (APC-Cy7® Conjugate)
Antibody Conjugates
Monoclonal Antibody

CD27 (O323) Mouse mAb (APC-Cy7® Conjugate) #19805

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Flow Cytometry Image 1 - CD27 (O323) Mouse mAb (APC-Cy7® Conjugate)

Flow cytometric analysis of live human peripheral blood mononuclear cells using CD27 (O323) Mouse mAb (PE-Cy7® Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (PE-Cy7® Conjugate) #79339 (dashed line).

To Purchase # 19805S
Product # Size Price
19805S
500 µl  (100 tests) $ 279

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG1 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 PE-Cy7® 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, pH7.2. 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 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

CD27 (O323) Mouse mAb (PE-Cy7® Conjugate) recognizes endogenous levels of total CD27 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

CD27 (TNFRSF7) is a transmemebrane protein of the TNF receptor superfamily (TNFRSF). It is mainly expressed on lymphoid cells (also on early hematopoietic precursor cells in mice) (1,2). CD27 is considered a phenotypic marker for memory B cells and is also used to identify B regulatory (Breg) cells (3,4). It is constitutively expressed on naïve CD4 and CD8 T cells and its expression is further upregulated upon T cell activation. CD27 is one of the two most important co-stimulatory receptors for T cell priming (the other one is CD28). While CD28 co-stimulatory signal mainly triggers cell proliferation, CD27 co-stimulatory signal primarily promotes cell survival and differentiation (5,6). Upon binding to its ligand CD70, CD27 activates the NF-κB and JNK signaling pathways through TNFR associated factors (TRAFs), the adaptor molecules that are associated with CD27 cytoplasmic tail domain. Upon activation CD27 is shed from cell surface and soluble CD27 is used as a marker of T cell activation (7,8).

  1. So, T. et al. (2006) Int J Hematol 83, 1-11.
  2. Waight, J.D. et al. (2017) Hum Antibodies 25, 87-109.
  3. Agematsu, K. (2000) Histol Histopathol 15, 573-6.
  4. Bouaziz, J.D. et al. (2012) Curr Mol Med 12, 519-27.
  5. Croft, M. Cytokine Growth Factor Rev 14, 265-73.
  6. Acuto, O. and Michel, F. (2003) Nat Rev Immunol 3, 939-51.
  7. Lens, S.M. et al. (1998) Semin Immunol 10, 491-9.
  8. van de Ven, K. and Borst, J. (2015) Immunotherapy 7, 655-67.

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.
Cy and CyDye are registered trademarks of GE Healthcare.