MHC Class II I-Ak Mouse Monoclonal Antibody [Clone ID: 14V.18]

CAT#: SM083B

MHC Class II I-Ak mouse monoclonal antibody, clone 14V.18, Biotin

Product Images

Picture 2

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  Picture 1

Specifications

Product Data

• Clone Name: 14V.18
• Applications: Assay, FC
• Recommended Dilution: Flow Cytomtry.
• Reactivities: Mouse
• Host: Mouse
• Isotype: IgG2a
• Clonality: Monoclonal
• Immunogen: A.TL spleen
  Donor: A.TH
  Fusion Partner: P3-X63-Ag 8
• Specificity: This monoclonal antibody is a cytotoxic antibody specific for cells expressing the Ia antigen coded for by the A subregion of the k haplotype. The reaction pattern of this antibody with a panel of inbred and recombinant haplotypes demonstrates that the antibody reacts with Ia.m2, a private specificity of the H-2k haplotype. This antibody can be used to quantitate or to eliminate cells bearing the I-Ak (Ia.m2) antigen.
• 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 G Chromatography
• Conjugation: Biotin
• Synonyms: H2-Aa
• 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®-M 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 1.0 - 0.5 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.
  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:
  Mouse Strain: A.TL
  Cell Concentration: 1x10e6 cells per tests
  Antibody Concentration Used: 0.5 µg/10e6 cells
  Isotypic Control: Biotin Mouse IgG2a
  
  Cell Source Percentage of cells stained above control:
  Thymus: 23.6%
  Spleen: 63.9%
  Lymph Node: 32.1%
  Bone Marrow: 11.1%
  
  Results - Strain Distribution:
  Antibody Concentration Used: 1.0 µg/10e6 cells
  Strains Tested: see Picture 1