MHC Class II I-Ek Mouse Monoclonal Antibody [Clone ID: 14-4-4S]

CAT#: CL074B

MHC Class II I-Ek mouse monoclonal antibody, clone 14-4-4S, Biotin

Product Images

Figure 1

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  Figure 2

Specifications

Product Data

• Clone Name: 14-4-4S
• Applications: Assay, FC
• Recommended Dilution: Flow Cytometry.
• Reactivities: Mouse
• Host: Mouse
• Isotype: IgG2a
• Clonality: Monoclonal
• Immunogen: C3H
  Donor: C3H.SW
  Fusion Partner: SP2/0-Ag 14
• Specificity: This monoclonal antibody is specific for cells expressing the Ia antigen coded for by the Ea subregion. The reaction pattern of this antibody with a panel of standard and recombinant haplotypes demonstrates that this antibody reacts with antigen Ia.m7, which is expressed by all haplotypes except b,f,q and s. This antibody is well suited for the quantitation and elimination of Ia bearing cells.
• 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
• 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 0.2-0.5 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 by Flow Cytometry Analysis:
  Mouse Strain: CBA/J
  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:
  Spleen: 53.4% see FIGURE 1
  Lymph Node: 16.5%
  Bone Marrow: 25.6%
  
  Strain Distribution by Flow Cytometry Analysis:
  Antibody Concentration Used: 0.5 µg/10e6 cells
  Strains Tested: see FIGURE 2; For a more detailed strain distribution - see reference 1.