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

Specifications

Product Data
Clone Name	14-4-4S
Applications	FC
Recommend Dilution	Flow Cytometry.
Reactivity	Mouse
Host	Mouse
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 can be used to quantitate or eliminate cells bearing the Ia.m7 antigen and is well suited for identifying Ia cell populations of positive mouse strains.
Formulation	PBS, 0.02% NaN3 and EIA grade BSA as a stabilizing protein to bring total protein concentration to 4-5 mg/ml. Label: FITC State: Liquid purified
Concentration	lot specific
Purification	Protein G Chromatography
Conjugation	FITC
Storage Condition	Store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer. Avoid repeated freezing and thawing. This product is photosensitive and should protected from light.
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. (It is recommmended that the tubes are protected from light, since most flurochromes are light sensitive.) 7. Wash 2 times at 4°C. 8. Resuspend the cell pellet in 50 µl ice cold media B. 9. 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: 0.2 µg/10e6 cells Isotypic Control: FITC Mouse IgG2a Cell Source Percentage of cells stained above control: Spleen: 53.5% see FIGURE 1 Lymph Node: 26.6% Bone Marrow: 38.9% Strain Distribution by Flow Cytometry Analysis: Procedure: As above Antibody Concentration: 0.2 µg/106 cells Strains Tested: see FIGURE 2; For a more detailed strain distribution - see reference 1.
Reference Data

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