Index. Index 425. Bacteria flow cytometry, general considerations, 33, 34 Gram-negative versus Grampositive

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1 Index 425 Index Acridine orange, see DNA/RNA simultaneous analysis Aerosol management system (AMS), biohazardous material sorting, 421 AMS, see Aerosol management system Annexin V, see Apoptosis, flow cytometry assays Antibodies, combining strategies, 63, 64 fluorochrome conjugation, 46 Apoptosis, flow cytometry assays, combination assay for detection of caspase activation, phosphatidylserine flipping, and cell permeability, annexin V labeling, 147, 156 caspase substrate labeling, 147, 156 cell preparation, 146, 147 data acquisition, 148 DNA-binding dye labeling, 148 gating, 150, 152 instrumentation, 145, 146 sample results and pleiotrophy, , 157, 158 simultaneous immunophenotyping, 152, 156, 157 cytotoxic T-lymphocyte assay of target cell killing using flow cytometry caspase substrates, advantages, 127, 128 caspase substrates for target cell apoptosis detection, , 134 chromium-51 release assay limitations, 126, 127 data analysis, 130, effector cell preparation, 129, 131 flow cytometry, 130, 132, 136 incubation conditions, 131, 132, 137 materials, , 134, 136 signaling of target cell killing, 125, 126 target cell preparation, , 134, 137 materials, 142, 143, 145, 155 overview, 141, 142 Autofluorescence, suppression technique, Bacteria flow cytometry, general considerations, 33, 34 Gram-negative versus Grampositive typing, data collection, 38 materials, 37, 38 overview, 36, 37 performance, 38, 39 sample preparation, 38, 42 viability assays, 34 36, 39, 40, 42 Biohazard, see Biosafety level-3 facility cell sorting Biosafety level-3 facility cell sorting, aerosol management system, 421 equipment,

2 426 Index overview, 419, 420 personal safety equipment, reagents and supplies, 420, 421 tolerances and measurement of containment, 421, 422, 424 Bivariate histogram, data display, 51 53, 55 Bromodeoxyuridine, see Cell cycle analysis Caspases, combination flow cytometry apoptosis assay for detection of caspase activation, phosphatidylserine flipping, and cell permeability, annexin V labeling, 147, 156 caspase substrate labeling, 147, 156 cell preparation, 146, 147 data acquisition, 148 DNA-binding dye labeling, 148 gating, 150, 152 instrumentation, 145, 146 sample results and pleiotrophy, , 157, 158 simultaneous immunophenotyping, 152, 156, 157 cytotoxic T-lymphocyte assay of target cell killing using flow cytometry caspase substrates, advantages, 127, 128 caspase substrates for target cell apoptosis detection, , 134 chromium-51 release assay limitations, 126, 127 data analysis, 130, effector cell preparation, 129, 131 flow cytometry, 130, 132, 136 incubation conditions, 131, 132, 137 materials, , 134, 136 signaling of target cell killing, 125, 126 target cell preparation, , 134, 137 CD34-negative precursor, see Hematopoietic stem cells Cell cycle analysis, bromodeoxyuridine treatment of cell culture, 346, 347, 351 flow cytometry, data analysis, data collection, 347, 352 Hoechst staining, 346 materials, 346, 347 principles of asynchronous population analysis, 345, 346 propidium iodide staining, 346, 347, 351, 352 sampling of cells, 347, 351 tumors, 359, 363 CFC, see Cytokine flow cytometry Cytokine flow cytometry (CFC), activation of cells, 99, 103 blood sample collection and preparation, 99, 102, 103 combination surface marker, cytokine, and intracellular phospho-staining, cell preparation, 86 fixation and permeabilization, 86 intracellular staining, 86 live/dead discrimination, 85, 91 materials, 73 overview, 85, 90, 91 surface staining, 86

3 Index 427 data acquisition and analysis, 101, 102, 104 historical perspective, 95, 96 materials, activation antigens, 98 equipment, 97 reagents, 97 principles, 96, 97 sample processing, peripheral blood mononuclear cells, 100, 101, 104 whole blood, 99, 100, 103, 104 T cells, 114, 117, 118 Cytotoxic T lymphocyte, see T cell Diode lasers, see Lasers Discosoma coral red fluorescent protein (DsRed), cell line expression, cell lines, 221 electroporation, 240, 242, 257 materials, 220, 221, 229, 231 retroviral transduction, 221, 222, 231 simultaneous detection of multiple fluorescent proteins, autofluorescence suppression, cell growth effects on signal strength compensation, fluorescence intensity distribution over cell cycle, 251, 253, 254 signal stability over time, 248 data acquisition and analysis, 224, 233 data display, 224, 225, 233, 234 detection optics and schemes, 229, 234 FACSVantage SE/FACSDiVa settings, 224, 233 heterogeneity suppression in detection, 244, 245, 247, 248 instrument configuration, 222, 223, 231 laser configurations, laser types, 242 one- or two-laser excitation, 242, 243, 257 three-laser excitation, 243, 254 overview, 219 spectral characteristics of proteins, 225, 229, 244 spectral characteristics, 220 DNA microarray, flow cytometry integration, Arabidopsis thaliana oligonucleotide microarrays, immobilization of oligonucleotide array, 269, 275 oligonucleotide preparations for printing, 268, 269 rehydration and printing, 269, 275 cell sorting of complex tissues, 260, 261 fluorescent protein tagging of specific cell types, materials, 267, 268 rationale, 259, 260 RNA labeling, overview, 272 poly(a) RNA, 273 total RNA, 273 RNA preparation, hybridization, 274, 275 overview, 269, 275, 276 poly(a) RNA, 271, 272 target purification, 273, 274, 276 total RNA, 270, 271 principles, 263, 265, 266

4 428 Index RNA amplification techniques, 266, 267 DNA/RNA simultaneous analysis, acridine orange, characteristics, 371, 372 staining, 373, 379 applications, lymphoreticular malignancies, 377 solid tumor analysis, 377, 379, 382 flow cytometry instrumentation and data acquisition, 373, 374 histogram analysis, 374, 377 materials, 372, 373 reproducibility, 377, 382 RNA content analysis, 377 standards, normal peripheral blood lymphocytes, 374 RNA content, 374 DsRed, see Discosoma coral red fluorescent protein Flow cytometer, analog-to-digital converters, analysis point, 5 7 application versatility, 27, 28, 27 color gating, 55 58, 63 data, analysis, 18 20, 22 bivariate histogram display, 51 53, 55 list mode file, 46, 47 output, 17, 18 phenogram analysis, univariate histogram display, 48, 49 definition, 2 fluidics, 7 10 fluorescence sources for detection, 3, 4 gating, 20, 22, historical perspective, 1, 2, 22, 25, 26 lasers, 4, 5 performance evaluation, 47, 48 signal amplification, 16, 17 signal detection, fluorescent light, 12 14, 45 scattered light, 10 12, 45, 46 sorting, mechanism, parameters, 25, 26 spatial resolution of particles, 3 Flow-fluorescence in situ hybridization, see Telomere length Fluorescence in situ hybridization (FISH), see Telomere length Fluorescence resonance energy transfer (FRET), flow cytometry studies of tumor necrosis factor-α/traf2 interactions, cell harvesting, 287 construct expression testing, fluorescence monitoring, 286 transfection, 285, 290 Western blot, 285 data acquisition, 287, 290 data analysis, materials, 283, 284 rationale, 283 transfections, 286, 287, 290 vector generation, 285, 290 green fluorescent protein donor/ acceptor pairs, 282, 283 human immunodeficiency virus virion fusion assay, flow cytometry data acquisition and analysis, 339, 341 immunostaining and fixation, 338, 339, 342 materials, overview, 333, 334, 341 target cell incubation with virions, 337

5 Index 429 target cell loading with substrate and BlaM reaction development, 338 virion production containing BlaM-Vpr, Gag enzyme-linked immunosorbent assay, 337 transfection, 336, 337, 341 viral supernatant harvesting and concentration, 337, 342 principles, 282 FRET, see Fluorescence resonance energy transfer Gating, color gating, 55 58, 63 principles, 18 20, Gene expression, see DNA microarray GFP, see Green fluorescent protein Green fluorescent protein (GFP), cell line expression, cell lines, 221 electroporation, 240, 242, 257 materials, 220, 221, 229, 231 retroviral transduction, 221, 222, 231 fluorescence resonance energy transfer donor/acceptor pairs, 282, 283 simultaneous detection of multiple fluorescent proteins, autofluorescence suppression, cell growth effects on signal strength compensation, fluorescence intensity distribution over cell cycle, 251, 253, 254 signal stability over time, 248 data acquisition and analysis, 224, 233 data display, 224, 225, 233, 234 detection optics and schemes, 229, 234 FACSVantage SE/FACSDiVa settings, 224, 233 heterogeneity suppression in detection, 244, 245, 247, 248 instrument configuration, 222, 223, 231 laser configurations, laser types, 242 one- or two-laser excitation, 242, 243, 257 three-laser excitation, 243, 254 overview, 219 spectral characteristics of proteins, 225, 229, 244 tagging of specific cell types in flow cytometry/dna microarray analysis, variants, 219, 220, 262 Helium neon lasers, see Lasers Hematopoietic stem cells, see also Side population assay, CD34-negative precursor analysis, cell sorting, 205, 206, 210 immunomagnetic separation for depletion of LIN + cells, 204, 205 materials, mobilized peripheral blood cell culture, 202 rationale, 201, 202 staining for immunophenotype and cell proliferation status, CD34, CD38, LIN, and Ki67 staining, 208 CD34, CD38, LIN, and PY staining, 209, 210, 216 cell activation and antigen modulation analysis, 210, 215, 216 overview, 206

6 430 Index thawed cell preparation, 204, 215 Hoechst identification, overview, 162 side populations, see Side population assay Hoechst 33342/rhodamine 123 staining assay, ABC transporters in dye efflux, 182, 183 applications, 181, 182 bone marrow cell harvesting, 187, 188, 195 Ho dull /Rh dull cells, cell sorting, 193, 196 relationship to side population cells, 183, 184 sort gating strategies for isolation, 191, 192, 196 instrument settings, 190, 191 limitations in dye-based cell sorting, 184, 185 low-density cell preparation, 188, 195 materials, , negative immunomagnetic selection, 188, 189, 195, 196 principles, 182, 183 staining, 190, 196 HIV, see Human immunodeficiency virus Ho dull /Rh dull cells, see Hematopoietic stem cells Hoechst 33258, see Cell cycle analysis Hoechst 33342, see Hematopoietic stem cells; Side population assay Human immunodeficiency virus (HIV), virion fusion fluorescence resonance energy transfer assay, flow cytometry data acquisition and analysis, 339, 341 immunostaining and fixation, 338, 339, 342 materials, overview, 333, 334, 341 target cell incubation with virions, 337 target cell loading with substrate and BlaM reaction development, 338 virion production containing BlaM- Vpr, Gag enzyme-linked immunosorbent assay, 337 transfection, 336, 337, 341 viral supernatant harvesting and concentration, 337, 342 Immunophenotyping, see Hematopoietic stem cells; Kinase cascade flow cytometry; Leukocyte flow cytometry; T cell; Telomere length JAK-STAT signaling, see Mammalian protein protein interaction trap Ki67, see Hematopoietic stem cells Kinase cascade flow cytometry, adherent cell assays, 92 advantages, 67, 68 cell lines, 70 combination surface marker, cytokine, and intracellular phospho-staining, cell preparation, 86 fixation and permeabilization, 86 intracellular staining, 86 live/dead discrimination, 85, 91 materials, 73 overview, 85, 90, 91 surface staining, 86 data analysis, 87 experimental parameters, 70

7 Index 431 methanol rehydration protocol for surface plus intracellular staining, cell preparation, fixation, and permeabilization, 82 materials, 72, 73, 87, 89 single-step surface marker staining, 84, 90 staining, 82, 84 phospho-specific antibodies, 68, 69 phospho-specificity testing in absence of pharmacological agents, 91, 92 sample preparation, 69 saponin protocol for surface plus intracellular staining, cell preparation, 84 fixation and permeabilization, 84 materials, 73, 89 staining, 85 surface marker, intracellular phospho-staining, and intracellular protein staining, 85 single phospho-staining, cell preparation, 74, 75, 89 fixation and permeabilization, 75, 89 materials, 72, 73, 87, 89 multiple kinase staining, 76, 77 staining, 75, 76, 89, 90 surface marker and intracellular phospho-staining, 77, 79, 90 Lasers, flow cytometry, air-cooled gas lasers, 400 charge-coupled device beam profiling system, 404, 415 configurations for multiple fluorescent protein detection, laser types, 242 one- or two-laser excitation, 242, 243, 257 three-laser excitation, 243, 254 diode lasers, cells and fluorochromes for flow cytometer analysis, 409, 410 overview, 400, 401 diode-pumped solid-state lasers, 401 helium neon green 594-nm laser, advantages, 401 cells and fluorochromes for flow cytometer analysis, 409 flow cytometer performance analysis, 410, 415 manufacturers, 402, 415 helium neon yellow 592-nm laser, advantages, 401 cells and fluorochromes for flow cytometer analysis, 409 flow cytometer performance analysis, 410, 411 manufacturers, 402 near-ultraviolet diode lasers, flow cytometer performance analysis, 413 manufacturers, 403 overview, 402 overview, 4, 5 power meters, 404 violet diode lasers, flow cytometer performance analysis, 411, 413 manufacturers, 403 overview, 402 water-cooled gas lasers, advantages and limitations, 399 Leukocyte flow cytometry, see also T cell, antibody combinations, 63, 64 data collection, 46

8 432 Index data display, bivariate distributions, 51 53, 55 color gating, 55 58, 63 gating, phenogram analysis, 59 61, 63 univariate histograms, 48, 49 fluorochromes, 46 instrument performance evaluation, 47, 48 materials, 47 LIN, see Hematopoietic stem cells Mammalian protein protein interaction trap (MAPPIT), fluorescence-activated cell sortingbased screening, expression vectors for receptor bait chimera, 299 fluorescence-activated cell sorting, 305, 308 host cell line, 298, 299, 307 isolated cell analysis, functional assay for interacting proteins, 305 prey identification, 305, magnetic cell sorting presort, 305 materials, overview, 296 prey cdna libraries, 299, 300, 302 retroviral infection and stimulation, 304, 308 screening condition determination, , 308 stable expression of receptor bait chimera, 302, 307, 308 principles, 293, 294 MAPPIT, see Mammalian protein protein interaction trap Membrane permeability, bacteria viability assays, 34 36, 39, 40, 42 Membrane potential, bacteria viability assays, 34 36, 39, 40, 42 Microarray, see DNA microarray Near-ultraviolet diode laser, see Lasers Nucleic acid analysis, see DNA/RNA simultaneous analysis P-glycoprotein, see Hematopoietic stem cells; Side population assay Phenogram, data analysis, Phosphatidylserine, see Apoptosis, flow cytometry assays Phosphoproteins, see Kinase cascade flow cytometry Ploidy, see Tumor DNA content analysis Protein protein interactions, see Fluorescence resonance energy transfer; Mammalian protein protein interaction trap PY, see Hematopoietic stem cells Rhodamine 123, see Hematopoietic stem cells RNA analysis, see DNA/RNA simultaneous analysis scfv, see Yeast scfv surface display library Senescence, see Telomere length Side population (SP) assay, flow cytometry, 166, 167, 176 Ho dull /Rh dull cell relationship to side population cells, 183, 184 Hoechst staining, 166, 167, 174, 176 human cell heterogeneity, 162, 163 human cord blood mononuclear cell isolation, 165, 166, 172, 174

9 Index 433 immunophenotyping and functional characterization, engraftment of human cord blood SP cells in NOD/ SCID mice, 169, 171 Hoechst efflux assay, 171, 177 mouse bone marrow cell surface antigens, 167, 169 materials, 163, 164, 171, 173 mouse bone marrow cell isolation, 165, 173, 174 P-glycoprotein role in phenotype, 162 SP assay, see Side population assay T cell, antigen specificity, 109, 110 cytokine flow cytometry, 95 cytotoxic T-lymphocyte assay using flow cytometry, advantages, 127, 128 caspase substrates for target cell apoptosis detection, , 134 chromium-51 release assay limitations, 126, 127 data analysis, 130, effector cell preparation, 129, 131 flow cytometry, 130, 132, 136 incubation conditions, 131, 132, 137 materials, , 134, 136 signaling of target cell killing, 125, 126 target cell preparation, , 134, 137 proliferation precursor frequency determination using celltracking dyes, cell culture, 116, 117, 122 cell culture treatment for cytokine retention, 113, 117 conventional assays, 110, 121 data analysis, , 123, 124 flow cytometry, 118, 123 materials, 111, 113, 114 overview, staining, CFSE, 116, 122 cytokines, 114, 117, 118 PKH dyes, 115, 116, 122 surface phenotype, 113, 114, 117, 122 tetramers, 113, 114, 117, 122 tracking dye for cell culture, 111, , 122 telomere length measurement using flow-fluorescence in situ hybridization, 392, 393 Telomere length, flow-fluorescence in situ hybridization, advantages, 386 controls, 390, 396 data analysis, 391, 392 flow cytometry, 391, 396 materials, 386, 387, 395 sample preparation, 390, 391, 396 simultaneous immunophenotyping, antibody cross-linking, 394, 395 applications, 395 cell surface labeling, 394 flow cytometry and data analysis, 395 materials, 394, 396, 397 overview, 393, 394 standardization and calibration, bead calibration of flow cytometer, , 396 conversion line calibration, 389, 390 T-cell analysis, 392, 393

10 434 Index shortening in senescence, 385, 386 telomerase in maintenance, 385, 386 TNF-α, see Tumor necrosis factor-α TRAF2, see Fluorescence resonance energy transfer Tumor DNA content analysis, see also DNA/RNA simultaneous analysis, controls, 359, 363 data analysis, cell cycle, 359, 363 ploidy, 359, 361, 366, 367 flow cytometry instrumentation and data acquisition, 359, 365 fresh cell preparation and propidium iodide staining, 357, 358, 363 overview, 355, 356 paraffin-embedded tissue sample preparation, 358, 359, solutions, archived samples, 357 fresh samples, 356 Tumor necrosis factor-α (TNF-α), see Fluorescence resonance energy transfer Univariate histogram, data display, 48, 49 Violet diode laser, see Lasers Yeast scfv surface display library, cell growth and induction, individual clones, 319 libraries, 318, 319, 331 clone validation and affinity determination, dissociation constant determination, 325, 327, 328 flow cytometric staining of individual clones, 325 materials, antigen biotinylation, 317 flow cytometry and magnetic bead enrichment, 317, 318 media and agar plates, 315, 317, 331 yeast strains and plasmids, 317 mutagenic libraries, 314 principles, 311, 312, 314 selection using magnetic bead enrichment and fluorescence-activated cell sorting, enrichment factor by selection round, 316 fluorescent staining of cells, 320, 321, 331 magnetic bead enrichment, 319, 320 overview, 314 sorting of clones from nonimmune library, first sort, 321, 322, 331 second sort, sort gate decision, 321 sorting for isolation of higher affinity clones, 329, 330 troubleshooting, 330, 331