MHC Class I H-2 Kb / H-2 Db Mouse Monoclonal Antibody [Clone ID: 5041.16.1]

Specifications

Product Data
Clone Name	5041.16.1
Applications	FC
Recommend Dilution	Flow Cytometry (see Protocol below).
Reactivity	Mouse
Host	Mouse
Clonality	Monoclonal
Specificity	This antibody reacts with cells expressing the H-2K antigen coded for by the b haplotype and for cells expressing the H-2D antigen coded for by the b haplotype. Results by Flow Cytometry Analysis: -Tissue distribution Mouse Strain: C57BL/6 Cell concentration: 1x10e6 cells per test Antibody concentration used: 0.1 µg/10e6 cells Isotypic control: Biotin Mouse IgG2a,k Cell source thymus: Percentage of cells stained above control = 35.5% Cell source spleen: Percentage of cells stained above control = 99.2% - Strain distribution Cell concentration : 1x10e6 cells per test Antibody concentration used: 0.1 µg/10e6 cells Strains tested: C57BL/6, BALB/c, C3H/He, CBA/J Positive: C57BL/6 Negative: BALB/c, C3H/He, CBA/J
Formulation	PBS containing 0.02% 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	lot specific
Purification	Affinity Chromatography on Protein A.
Conjugation	Biotin
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.
Synonyms	HLA Class I
Note	Protocol: FLOW CYTOMETRY ANALYSIS Method: 1. Prepare a cell suspension in media A. For spleen 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 0.1 µg of CL058B 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 appropriate 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).
Reference Data

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