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64776
MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (APC Conjugate)
Antibody Conjugates

MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (APC Conjugate) #64776

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Flow cytometric analysis of live mouse splenocytes using MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (APC Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (APC Conjugate) #34828 (dashed line).

To Purchase # 64776S
Product # Size Price
64776S
100 µg $ 219

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rat IgG2b, kappa

Product Description

This Cell Signaling Technology antibody is conjugated to allophycocyanin (APC) 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

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Immunostaining

  1. Count cells using a hemacytometer or alternative method.
  2. Aliquot 0.5-1x106 cells into each assay tube (by volume).
  3. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  4. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  5. Incubate for 1 hr at room temperature.
  6. Wash by centrifugation in 2-3 ml incubation buffer.
  7. Resuspend cells in the appropriate volume of PBS and analyze on flow cytometer.

posted June 2017

Protocol Id: 1504

Specificity / Sensitivity

MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (APC 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, and H-2u haplotypes, 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.

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

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