Flow cytometric analysis of live mouse splenocytes using MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (APC-Cy7® Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (APC-Cy7® Conjugate) #63801 (dashed line).
|Source/Isotype||Rat IgG2b kappa|
This Cell Signaling Technology antibody is conjugated to APC-Cy7® and tested in-house for direct flow cytometry analysis in mouse cells.
For optimal flow cytometry results, we recommend 0.5 μg of antibody per test. A slight precipitate may be present, but will not interfere with antibody performance. If precipitates are present, centrifuge the tube at 6,000xg for 10-30 sec. Draw off the supernatant and place into a light protective vial.
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
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.
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.
posted June 2017
revised June 2020
Protocol Id: 1504
MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (APC-Cy7® 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).
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.
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).
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