addition to the f1uorochromes used for labeling monoclonal antibodies, a wide variety of dyes with useful biological properties can be used to identify cell populations or measure functions in living cells (Table 6.1). 6.4 HOW THE CYTOMETER WORKS After cells have been labeled with dye-conjugated antibodies or other fluorescent molecules, they are resuspended at an appropriate concentration and introduced "single file" into the analytical cytometer. The cell sample is injected into a stream of sheath fluid (saline or distilled water), such that the sample stream forms a stable 186 Handbook of Human Immunology, Second Edition TABLE 6.1 Some Commonly Used Dyes and Fluorescent Molecules Dye DAPI [22-25] 7-AAD [26J Acridine orange [27-29] Aldefluor [30] BODIPY [31,32] CFSE [33] Draq5 [34] Green fluorescent protein (GFP) and its variants [35,36] Hoechst 33342 [23,37,38] Indo-I [39,40] JC-I [41,42] Monochlorobimane [43] Phi Phi Lux [44] Propidium iodide (PI) [45-48] Rhodamine 123 [37,49] Maximal Excitation/ Emission (nm) 358/~61 nm. bound to D A 546/647 495/519 505/513 505/513 494/514 647/670 (huge tail allows excitation with 488 line) 395/509 350/461 330/40 1,475 514/529 394/490 505/530 535/617 nm, bound to nucleic acids 507/529 Properties Bind, to AT-rich regions of D A. Viability staining (viable cells exclude dye): Nuclear (D A) staining in permeabilized cells Viability staining (viable cells exclude dye): Nuclear (DNA) staining in penneabilized cells Measurement of DNA and (ribonucleic acid) RNA Nonfluorescent molecule (BODlPYaminoacetaldehyde) that fluoresces when converted to BODIPY-aminoacetate by aldehyde dehydrogenase (active in stem cells) Labeling drugs for uptake and efflux studies Labeling cytoplasmic proteins of viable cells (cell tracking and proliferation assays) Nuclear (DNA) staining of living (nonpermeabilized) cells Identification and sorting of transfected cells. Variants provide alternative excitation/ emissions maxima Nuclear (DNA) staining of living (nonpermeabilized) cells; Measurement of DNA content (cell cycle); Detection of cells with constitutive MDR transporter activity (side population) Intracellular calcium concentration (ratiometric) Mitochondrial membrane potential Measurement of intracellular glutathione Quenched fluorescent molecule linked to the caspase-3 substrate amino acid sequence (DEVDGl). Fluoresces when cleaved by caspase-3 (detection of apoptosis) Viability staining (viable cells exclude dye); Nuclear (DNA) staining in permeabilized cells MDR transporter-mediated dye efflux, mitochondrial mass Note: DAPI--4,6-diamidinozphenylindole; CFSE-carboxy fluorescein succinimydal ester; 7AAD7-amino actinomycin-D; BODlPY-DlPYrromethene BOron Difluoride. core within the sheath stream. Because laminar flow is established between the two streams, sample and sheath fluid do not normally mix, and the cells are concentrated near the center of the sample stream by a process called hydrodynamic focusing. In an analytical cytometer, which does not have the additional task of sorting the cells, r Understanding Clinical Flow Cytometry 187 all this takes place within a quartz flow cell in which the cells are illuminated by one or more lasers. Light scattered by the cells is captured at a small angle (forward light scatter [FSc]) and at 90° (side scatter ISSc)) from the incident light as they cross the laser path (Figure 6.1). FSc correlates well with cell size. whereas SSc measures the cell's internal complexity (nuclear convolutions, vacuoles, etc.). Forward scattered light, because of its intensity, is usually detected by a relatively insensitive and inexpensive photodiode, whereas side scattered light, such as light emitted by the fluorochromes with which the cells are labeled, is measured using very sensitive PMTs. [n peripheral blood, small cellular debris, lymphocytes, granulocytes, and monocytes can be distinguished on the basis of their intrinsic light-scattering properties alone (Figure 6.2). The photons emitted by fluorescent dyes associated with the cell are detected by PMTs, which with the aid of a series of filters, limit the window of detection to the major emission region of particular fluorochromes (e.g., with FlTC, the intensity of