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78355
Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb (PE Conjugate)
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Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb (PE Conjugate) #78355

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Flow cytometric analysis of live mouse bone marrow cells using Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb (PE Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (PE Conjugate) #27426 (dashed line).

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To Purchase # 78355S
Product # Size Price
78355S		 100 µg $ 125

Custom Formulation

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rat IgG2b

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 0.06 μg of antibody per test.

Application Dilutions
Flow Cytometry 1:300

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

Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb (PE Conjugate) recognizes endogenous levels of total Ly-6G/Ly-6C (Gr-1) 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

The Ly-6 complex is a series of genes found on chromosome 15. These genes code for a number of different proteins that can be used as surface markers. The family members vary in their biologic expression and have been shown to be involved in cell signaling and cell adhesion (1). The structure of these proteins includes a motif known as the LU domain that has three loops comprised of disulfide bonds. These bonds are formed by 8 to 10 cysteines that can cause differences in the length of the loops as well as the sequences at each tip (2,4). There are 11 known Ly-6 genes on murine chromosome 15 that code for different proteins. These family members, excluding secreted Ly6/Plaur domain containing 1 coded by the Slurp1 gene, are attached to the cell surface by a GPI anchor near the C terminus. The structure of these proteins may play a role in transmembrane interactions, and downstream signaling cascades (1,2).

Ly-6 proteins have been widely used as differentiation markers on hematopoietic cells. The ability to isolate and express specific Ly-6 antibodies through hybridoma technology has allowed researchers to identify unique proteins (1). These proteins are expressed on subsets of immune cells at different stages of development, such as T cells, B cells, monocytes, granulocytes, and macrophages (1-5). 

The 1A8 clone is specific to Ly-6G and is widely used to identify mature granulocytes (3). The RB6-8C5 clone recognized both Ly-6G and Ly-6C, also known as Gr-1, and has been found to express on neutrophils, monocytes, dendritic cells, and T cells (2,3).

  1. Bamezai, A. Arch Immunol Ther Exp (Warsz) 52, 255-66.
  2. Lee, P.Y. et al. (2013) J Leukoc Biol 94, 585-94.
  3. Fleming, T.J. et al. (1993) J Immunol 151, 2399-408.
  4. Tsetlin, V. (1999) Eur J Biochem 264, 281-6.
  5. Pflugh, D.L. et al. (2000) J Immunol 165, 313-21.

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.