PE-labeled beads are commercially available in a kit, which contains one tube with a mixture of beads with four different predefined levels of PE. The PE-labeled antibodies used for staining of surface antigens on blood cells are at a 1:1 fluorochrome-to-antibody ratio. This allows determination of the ABC when beads are run under the same photomultiplier and compensation settings as blood cells (see Notes 2 and 3).
All samples were analyzed on a FACS Calibur (BD Biosciences) using the software CellQuest™. The monocytes and granulocytes were separated on the basis of their forward scatter and side scatter patterns, and the staining with CD14 was used to check the identification of the monocytes. At least 3000 monocytes were analyzed per sample. Additionally, 10,000 events in the granulocyte gate were counted. The PE fluorescence within the gates was measured as specific geometric mean fluorescence intensity of the whole population of cells (see Note 4). QuantiQuest is a quantitative calibration feature within the CellQuest acquisition software that calculates the linear function relating fluorescence to PE molecules. So with help of the Microsoft Excel "-spreadsheet, geometric mean fluorescence was converted into the term ABC.
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