MHC Class II I-Ap Mouse Monoclonal Antibody [Clone ID: 7-16.17]

Specifications

Product Data
Clone Name	7-16.17
Applications	FC
Recommended Dilution	Flow Cytometry.
Reactivities	Mouse
Host	Mouse
Isotype	IgG2a
Clonality	Monoclonal
Immunogen	B10.P Donor: BALB/c Fusion Partner: SP2/0
Specificity	This monoclonal antibody is a cytotoxic antibody which defines a public I-A antigen. This antibody reacts with I-A antigen from the following I-A haplotypes: I-Ap,k,q,r,s,b. Using recombinant strains, reactivity against the b haplotype has been localized to the Ab subregion. This antibody can be used to quantitate or eliminate I-A bearing cells or for precipitating I-A 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	lot specific
Purification	Protein G Chromatography
Conjugation	Biotin
Storage	Store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer. Avoid repeated freezing and thawing.
Stability	Shelf life: one year from despatch.
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 by Flow Cytometry Analysis: Mouse Strain: BDP 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: 36.5% Spleen: 51.5% Lymph Node: 16.3% Bone Marrow: 24.4% 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.
Reference Data