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67762
MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (PE Conjugate)
Antibody Conjugates
Monoclonal Antibody

MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (PE Conjugate) #67762

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Flow Cytometry Image 1: MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (PE Conjugate)

Flow cytometric analysis of live mouse splenocytes using MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (PE Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (PE Conjugate) #27426 (dashed line).

To Purchase # 67762S
Product # Size Price
67762S
100 µg $ 159

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rat IgG2b 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 phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in mouse cells.

Product Usage Information

For optimal flow cytometry results, we recommend 0.06 μg of antibody per test.

Application Dilution
Flow Cytometry 1:300

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

MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (PE Conjugate) recognizes endogenous levels of total MHC class II (I-A/I-E) proteins. This antibody detects epitopes within the extracellular domain of MHC class II (I-A/I-E).

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

Major histocompatibility complex class II (MHC class II) molecules are heterodimeric, transmembrane glycoproteins expressed on the surface of antigen-presenting cells such as macrophages, dendritic cells, and B cells. Expression can also be induced on other cell types through interferon-γ signaling (1). Prior to being displayed on the cell membrane, MHC class II molecules are loaded with exogenous peptide antigens approximately 15-24 amino acids in length that were derived from endocytosed extracellular proteins digested in the lysosome (2). Antigen-presentation through MHC class II is required for T cell activation during the immune response to extracellular pathogens (2). In humans, the MHC class II protein complex is encoded by the human leukocyte antigen gene complex (HLA). HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR (3).

The M5/114.15.2 antibody reacts with mouse MHC class II, both I-A and I-E subregion-encoded glycoproteins (I-Ab, I-Ad, I-Aq, I-Ed, I-Ek, not I-Af, I-Ak, or I-As). It detects a polymorphic determinant present on B cells, monocytes, macrophages, dendritic cells, and activated T lymphocytes from mice carrying the H-2b, H-2d, H-2q, H-2p, H-2r and H-2u haplotypes, but not from mice carrying the H-2s or H-2f haplotypes (4-7). The M5/114 mAb is reported to inhibit I-A-restricted T cell responses of the H-2b, H-2d, H-2q, H-2u but not H-2f, H-2k, or H-2s haplotypes (8,9).

  1. Ting, J.P. and Trowsdale, J. (2002) Cell 109 Suppl, S21-33.
  2. Cresswell, P. (1994) Annu Rev Immunol 12, 259-93.
  3. Karp, D.R. et al. (1990) J Exp Med 171, 615-28.
  4. Staehli, F. et al. (2012) J Immunol 188, 3820-8.
  5. Scarlett, U.K. et al. (2012) J Exp Med 209, 495-506.
  6. Anderson, M.S. and Miller, J. (1992) Proc Natl Acad Sci U S A 89, 2282-6.
  7. Miyazaki, T. et al. (1996) Cell 84, 531-41.
  8. Parra, D. et al. (2012) J Leukoc Biol 91, 525-36.
  9. Chen, M. et al. (2011) J Immunol 187, 5684-92.

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.