MHC Class II (I-Ab,d) Mouse Monoclonal Antibody [Clone ID: 25-9-17S]

CAT#: AM31877BT-N

MHC Class II (I-Ab,d) mouse monoclonal antibody, clone 25-9-17S, Biotin

Product Images

Figure 1. Cell Source: Spleen. Percentage of cells stained above control: 58.6%

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  Figure 2.

Specifications

Product Data

• Clone Name: 25-9-17S
• Applications: Assay, FC
• Recommended Dilution: Flow Cytometry (See Protocols).
• Host: Mouse
• Isotype: IgG2a
• Clonality: Monoclonal
• Immunogen: C3H.SW splenocytes.
  Donor: C3H lymphoid cells.
  Fusion Partner: Sp2/0-Ag14
• Specificity: This Monoclonal Antibody reacts with I-Ab and I-Ad antigens. Cross reaction with H-2p and H-2q was also found.
• Formulation: PBS containing 0.02% Sodium Azideas preservative and EIA grade BSA as a stabilizing protein to bring total protein concentration to 4-5 mg/ml
  Label: Biotin
  State: Liquid purified Ig fraction
• 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 106 cells, representing 1 test).
  4. To each tube, add 0.5-0.2 μg* of AM31877BT-N 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: C57BL/6
  Cell Concentration : 1x10e6 cells per test.
  Antibody Concentration Used: 0.5 μg/106 cells.
  Isotypic Control: Biotin Mouse IgG2a
  
  Cell Source Percentage of cells stained above control:
  Thymus: 17.5%
  Spleen: 58.6%
  Lymph Node: 31.2%
  
  Strain Distribution by Flow Cytometry Analysis:
  Antibody Concentration: 0.5 µg/106 cells.
  Strains Tested: See Figure 2.