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In FlowJo v10, we need to start with data from your calibration standards. The strict measurement being determined here is the molecules of equivalent fluorescence (MESF). Note: In the following example, we assume one bound antibody per molecule, which may not be true depending on antibody class, distance between molecules, and number of targeted epitopes on a given molecule. With the standard curve we derive a linear relationship between fluorescence intensity and number of molecules on a given cell. Considering that fluorescence intensity is correlated with molecules on the surface of the cell, can the relationship between the two be quantified? If so, how can we use that relationship to calculate the number of molecules on the surface of a cell in a given experiment?Īs with all indirect measurements, a standard curve must first be created using calibration standards (for example, cytometric bead arrays), to establish the relationship between the fluorescence intensity measurements and the antibody binding to its target molecule. Measuring the fluorescence intensity of cells and particles is routine and the basis of the vast majority of inquiry in flow cytometry.