Robinson's Current Protocols in Cytometry PDF

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To make the dot-plot axes read in units of FITC and PE fluorescence rather than green and yellow fluorescence, the amount of spectral overlap from each fluorochrome can be subtracted (Bagwell and Adams, 1993). The method used to accomplish the subtraction is instrument dependent; ask the manufacturer for details. Since the percentage of FITC fluorescence in the yellow detector is always a constant fraction of the amount of FITC fluorescence in the green detector, the same percentage of green FITC fluorescence can be subtracted from the yellow signal no matter what the FITC signal is.

4E had calibration values for the stained beads (upper four peaks in the histogram) of 4,201, 16,936, 37,466, and 65,797 fluorescein MESF (molecules of equivalent soluble fluorochrome). 49-µm-diameter fluorescent bead that is useful for checking or adjusting optical alignment. All fluorescence CVs were <2%. General Cautions for Using Particles in Standardization and Calibration There are two important factors to remember when using manufactured particles rather than cells in a flow cytometer. First, beads are not cells and do not necessarily scatter light as cells do.

1996; see Characterizing Particles for Calibration and Control of a Flow Cytometer). To ensure that the secondary calibrator on an instrument is reliable, however, the emission filters and the wavelength and intensity of the excitation light must remain unchanged. 2 and cautions given by Schwartz et al. (1996). ) Antibody binding capacity (ABC) Standardization, Calibration, and Control in Flow Cytometry A further step in immunofluorescence standardization and calibration is to express measurement results in terms of antibody binding capacity.

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Current Protocols in Cytometry by Robinson

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