MHC Class II I-Ab Mouse Monoclonal Antibody [Clone ID: 25-5-16S]

CAT#: SM080B

MHC Class II I-Ab mouse monoclonal antibody, clone 25-5-16S, Biotin

Product Images

Specifications

Product Data

• Clone Name: 25-5-16S
• Applications: FC
• Recommended Dilution: Flow Cytometry (see protocol below).
• Reactivities: Mouse
• Host: Mouse
• Isotype: IgM
• Clonality: Monoclonal
• Specificity: This antibody specifically reacts with the I-Ab encoded MHC Class II alloantigen expressed on mouse strains BXSB/Mp, C57BL/ 6, C57BL/10, LP/J, and 129.
  Tissue Distribution by Flow Cytometry Analysis:
  Representative Histogram
  Mouse Strain: C57BL/6
  Cell Concentration : 1x10e6 cells per test
  Antibody Concentration Used: 0.5 µg/10e6 cells
  Isotypic Control: Biotin Mouse IgM.
• Formulation: PBS containing 0.09% Sodium Azide 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
• Conjugation: Biotin
• Synonyms: H2-Eb1, H-2 class II histocompatibility antigen I-A beta chain
• Note: Protocol: FLOW CYTOMETRY ANALYSIS:
  Method:
  1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cell population.
  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 antibody 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).