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
Recommended Dilution	Flow Cytometry (see Protocol below).
Reactivities	Mouse
Host	Mouse
Isotype	IgG2a
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	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.
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