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Extra info for Current Protocols in Cytometry
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.
Current Protocols in Cytometry by Robinson