Leptin R (APC)

Leptin receptor, also known as OB-R, is a type I cytokine receptor family protein that functions as a receptor for Leptin, an adipocyte-specific hormone that regulates adipocyte tissue mass through hypothalmic effects on satiety and energy expenditure.\n\nPrinciple of the Test:\nWashed cells are incubated with the allophycocyanin-labeled monoclonal antibody, which binds to cells expressing Leptin R. Unbound allophycocyanin-conjugated antibody is then washed from the cells. Cells expressing Leptin R are fluorescently stained, with the intensity of staining directly proportional to the density of expression of Leptin R. Cell surface expression of Leptin R is determined by flow cytometric analysis using 620-650 nm wavelength laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 660-670nm.\n\nReagent Preparation:\nAllophycocyanin-conjugated mouse anti-human Leptin R: Use as is; no preparation necessary.\nHuman monocytes were stained with APC-conjugated anti-human Leptin R or isotype control.\n\nSample Preparation:\nPeripheral blood cells: \nWhole blood should be collected in evacuated tubes containing EDTA or heparin as the anticoagulant. Contaminating serum components should be removed by washing the cells three times in an isotonic phosphate buffer (supplemented with 0.5% BSA) by centrifugation at 500 x g for 5 minutes. Transfer 50il of packed cells to a 5 ml tube for staining with the monoclonal antibody. Whole blood will require lysis of RBC following the staining procedure.\n\nCell Cultures: \nContinuous cell lines or activated cell cultures should be centrifuged at 500 x g for 5 minutes and washed three times in an isotonic PBS buffer (supplemented with 0.5% BSA), as described above, to remove any residual growth factors that may be present in the culture medium. Cells should then be resuspended in the same buffer to a final concentration of 4 x 10e6 cells/ml and 25il of cells (1 x 10e5) transferred to a 5 ml tube for staining.\nNote: Adherent cell lines may require pretreatment with 0.5 mM EDTA to facilitate removal from substrate. Cells that require trypsinization to enable removal from substrate should be further incubated in medium for 6-10 hours on a rocker platform to enable regeneration of the receptors. The use of the rocker platform will prevent reattachment to the substrate.\n\nSample Staining:\n1) Cells should be Fc-blocked by treatment with 1ug of human IgG/10e5 cells for 15 minutes at room temperature prior to staining. Do not wash excess blocking IgG from this reaction.\n2) Transfer 25il of the Fc-blocked cells (1 x 105 cells) or 50il of packed whole blood to a 5 ml tube.\n3) Add 10il of APC-conjugated Leptin R reagent.\n4) Incubate for 30-45 minutes at 2-8 degrees C.\n5) Following this incubation, remove unreacted Leptin R reagent by washing the cells twice in 4 ml of the same PBS buffer (note: whole blood will require an RBC lysis step at this point using any commercially available lysing reagent, such as Whole Blood Lysing Kit).\n6) Finally, resuspend the cells in 200-400il of PBS buffer for final flow cytometric analysis.\n7) As a control for analysis, cells in a separate tube should be treated with APC-labeled mouse IgG2B antibody.\nThis procedure may need modification, depending upon final utilization.\n\nStorage:\nMay be stored at 4 degrees C before opening. DO NOT FREEZE! Stable at 4 degrees C as an undiluted liquid. Dilute only prior to immediate use. Stable for 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. Freezing APC conjugates will result in a substantial loss of activity. APC conjugates are sensitive to light.

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