MHC Class I (RT1Ac) Mouse Monoclonal Antibody [Clone ID: OX-27]

CAT#: CL129B

MHC Class I (RT1Ac) mouse monoclonal antibody, clone OX-27, Biotin

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Specifications

Product Data

• Clone Name: OX-27
• Applications: FC, IHC
• Recommended Dilution: Flow Cytometry.
  Immunohistochemistry using cryostat sections: (however, cross-reactivity with Lewis rats have been shown to occur in some instances).
• Reactivities: Rat
• Host: Mouse
• Isotype: IgG2a
• Clonality: Monoclonal
• Immunogen: Phytohaemagglutinin Blasts
  Donor: BALB/c spleen
  Fusion Partner: P3-NS1-1-Ag4 (NS1/1)
• Specificity: This Antibody recognizes a polymorphic determinant of the MHC class I antigen in the rat. (c and n haplotypes). This antibody is an excellent antibody for labelling cells of donor or host origin in bone marrow chimeras.
• Formulation: PBS, 0.02% NaN3 and EIA grade BSA as a stabilizing protein to bring total protein concentration to 4-5 mg/ml
  Label: Biotin
  State: Liquid purified Ig
• Concentration: 0.1 mg/ml
• Purification: Protein A Chromatography
• Conjugation: Biotin
• Background: MHC Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen. MHC class I antigens are heterodimers consisting of one alpha chain (44kDa) with beta 2 microglobulin (11.5 kDa). The antigen is expressed by all somatic cells at varying levels. MHC Class I molecules are expressed on most nucleated cells where they present endogenously synthesized antigenic peptides to CD8+ T lymphocytes, which are usually cytotoxic T cells. Fibroblasts or neurons however only show a low level of antigen.
• Synonyms: RT1-A3
• Note: Protocol: FLOW CYTOMETRY ANALYSIS:
  
  Method:
  1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cell population with Lympholyte®-Rat cell separation medium.
  2. Wash 2 times.
  3. Resuspend the cells to a concentration of 2x10e7 cells/ml in media A. Add 50 µl of this suspension to each tube (each tube will then contain 1 x 10e6 cells, representing 1 test).
  4. To each tube, add 0.5-1.0 µg* of this Ab per 10e6 cells.
  5. Vortex the tubes to ensure thorough mixing of antibody and cells.
  6. Incubate the tubes for 30 minutes at 4°C.
  7. Wash 2 times at 4°C.
  8. Add 100 µl of secondary antibody (Streptavidin-FITC) at a 1:500 dilution. Not recommended for use with a PE secondary.
  9. Incubate tubes at 4°C for 30 - 60 minutes (It is recommended that tubes are protected from light since most fluorochromes are light sensitive).
  10. Wash 2 times at 4°C.
  11. Resuspend the cell pellet in 50 µl ice cold media B.
  12. Transfer to suitable tubes for flow cytometric analysis containing 15 µl of propidium iodide at 0.5 mg/ml in PBS. This stains dead cells by intercalating in DNA.
  
  Media:
  A. Phosphate buffered saline (pH 7.2) + 5% normal serum of host species + sodium azide (100 µl of 2M sodium azide in 100 mls).
  B. Phosphate buffered saline (pH 7.2) + 0.5% Bovine serum albumin + sodium azide (100 µl of 2M sodium azide in 100 mls).
  
  Results - Tissue Distribution:
  Rat Strain: Brown Norway
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 1.0 µg/10e6 cells
  Isotypic Control: Biotin Mouse IgG2a
  
  Cell Source Percentage of cells stained above control:
  Thymus 65.4%
  Spleen 98.9%
  Lymph Node 100%
  
  Restults - Strain Distribution:
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 1.0 µg/10e6 cells
  Strains Tested: Brown Norway, Buffalo, Wistar, Fischer, ACI
  Positive: Brown Norway
  Negative: Buffalo, Wistar, Fischer, ACI