INTRODUCTION TO FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 8

FIGURE 1.4 Excitation and emission spectra of FITC and phycoerythrin (PE). Fluorescent
molecules absorb light of a characteristic wavelength and emit light of a longer wavelength.
FITC and PE that are commonly used for flow cytometry absorb at 488 and 488-560 nm,
respectively, but emit at 520 and 590 nm, respectively. Thus, they can be excited by the same
laser line and used together in the same tube [10].
argon laser but emits in the 520 nm (green) range) that recognizes and binds to CD4,
the more the 520 nm emission the more the CD4 on the cell (Figure 1.4).
The fluorochrome label for a reagent depends on instrument configuration (type
and number of lasers and type of optical filters and detectors), which determines if a
given instrument can excite a given fluorochrome and detect the emission. While it is
not possible to uniformly state the best fluorochrome combination, there are a few
guidelines that can help in this choice. The first issue is to determine what is
the reagent brightness, which takes into account the resolvable signal associated
with the presence of the marker being detected by comparing a negative and a positive
sample. The negative population emission is the background emission. Background is
signal (emission) due to electronic noise (dark current), cell autofluorescence,
nonspecific staining, and background emission that is a spillover from another
fluorochrome [61, 62]. The rule of thumb is to use the brightest reagents possible [63,
64]. There is a caveat to this statement. The spillover problems increase as the number
of colors to be resolved (different emissions) increases. Compensation can help prevent
the spillover contribution, but as a rule of thumb, one should use fluorochromes whose
emissions have the least amount of spectral overlap [65, 66]. In addition, logically the