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60712
CD86/B7-2 (GL-1) Rat mAb (PE Conjugate)
Antibody Conjugates

CD86/B7-2 (GL-1) Rat mAb (PE Conjugate) #60712

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Flow cytometric analysis of live mouse splenocytes, untreated (left column) or treated with LPS #14011 (500 ng/ml, 3 d; right column), using CD86/B7-2 (GL-1) Rat mAb (PE Conjugate) (top row) or concentration-matched Rat Isotype Control (PE Conjugate) (bottom row), and co-stained with CD19 (ID3) Rat mAb (APC Conjugate) #39831.

To Purchase # 60712S
Product # Size Price
60712S
100 µg  (10 western blots) $ 189

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rat IgG2a, 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 and tested in-house for direct flow cytometric analysis in mouse cells.

Product Usage Information

For optimal flow cytometry results, we recommend 0.125 μg of antibody per test.

Application Dilution
Flow Cytometry 1:160

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

CD86/B7-2 (GL-1) Rat mAb (PE Conjugate) recognizes endogenous levels of total CD86/B7-2 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

CD80 (B7-1, BB1) and CD86 (B7-2, B70) are members of the B7 family of cell surface ligands that regulate T cell activation and immune responses. CD80 is expressed on activated antigen presenting cells, including dendritic cells, B cells, monocytes, and macrophages. CD86 is expressed on resting monocytes, dendritic cells, activated B lymphocytes, and can be further upregulated in the presence of inflammation (1-3). CD80 and CD86 are ligands for CD28, which functions as a T cell costimulatory receptor. Interaction of CD28 with CD80 or CD86 provides the second signal required for naïve T cell activation, T cell proliferation, and acquisition of effector functions (3-7). Alternatively, CD80 and CD86 also act as ligands to CTLA-4, which results in the downregulation of T cell activity (3,7-9).

The GL-1 antibody is used as a marker for CD86 expression on B cells, macrophages, and dendritic cells.

  1. Freeman, G.J. et al. (1989) J Immunol 143, 2714-22.
  2. Azuma, M. et al. (1993) Nature 366, 76-9.
  3. Adams, A.B. et al. (2016) J Immunol 197, 2045-50.
  4. Linsley, P.S. et al. (1990) Proc Natl Acad Sci U S A 87, 5031-5.
  5. Gimmi, C.D. et al. (1991) Proc Natl Acad Sci U S A 88, 6575-9.
  6. Harding, F.A. et al. (1992) Nature 356, 607-9.
  7. Collins, A.V. et al. (2002) Immunity 17, 201-10.
  8. Linsley, P.S. et al. (1991) J Exp Med 174, 561-9.
  9. Krummel, M.F. and Allison, J.P. (1995) J Exp Med 182, 459-65.

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.