Essential Cytometry Methods

1. II. Introduction 2. III. Labeling of Proteins with Organic Fluorescent Dyes 3. IV. Labeling of Antibodies with Zenon Probes 4. V. Labeling of Proteins with Phycobiliproteins 5. VI. Conclusion 6. Chapter 3. Cytometry of Fluorescence Resonance Energy Transfer 7. I. Introduction 8. II. Theory of FRET 9. III. Measuring FRET 10. IV. Applications 11. V. Perspectives 12. VI. Conclusions 13. Chapter 4. The Rainbow of Fluorescent Proteins 14. I. Update 15. II. The Fluorescent Proteins 16. III. Flow Analysis Using Fluorescent Proteins 17. IV. Flow Sorting Using Fluorescent Proteins 18. V. Conclusion 19. Chapter 5. Labeling Cellular Targets with Semiconductor Quantum Dot Conjugates 20. I. Introduction 21. II. Selection of QDs and their Conjugates 22. III. Labeling of Fixed Cells for Fluorescence Microscopy 23. IV. Measurements in Living Cells Using QD Conjugates 24. V. Conclusion 25. Chapter 6. High-Gradient Magnetic Cell Sorting 26. I. Update 27. II. Introduction 28. III. Application 29. IV. Materials 30. V. MACS: Staining and Sorting 31. VI. Critical Aspects of the Procedure 32. VII. Controls 33. VIII. Instruments 34. IX. Results 35. Chapter 7. Multiplexed Microsphere Assays (MMAs) for Protein and DNA Binding Reactions 36. I. Update 37. II. Introduction 38. III. Technology and Instrumentation 39. IV. Methods 40. V. Results 41. VI. Software 42. VII. Critical Aspects of the Methodology 43. VIII. Comparison with Other Methods 44. IX. Future Directions 45. Chapter 8. Biohazard Sorting 46. I. Recent Developments 47. II. Introduction 48. III. Critical Aspects of the Procedure 49. IV. Applications and Future Directions 50. Chapter 9. Guidelines for the Presentation of Flow Cytometric Data 51. I. Introduction 52. II. General Principles of Graphical Presentation 53. III. Statistics 54. IV. Subset Analysis 55. V. Conclusion 56. Chapter 10. Detergent and Proteolytic Enzyme-Based Techniques for Nuclear Isolation and DNA Content Analysis 57. I. Introduction 58. II. Basic Principles of the Methods 59. III. Applications 60. IV. Materials 61. V. Methods 62. VI. Results 63. Chapter 11. DNA Content and Cell Cycle Analysis by the Propidium Iodide-Hypotonic Citrate Method 64. I. Introduction 65. II. Application 66. III. Materials 67. IV. Staining Procedure 68. V. Caution 69. VI. Instrument Setup 70. VII. Comments 71. VIII. Results 72. Chapter 12. DNA Analysis from Paraffin-Embedded Blocks 73. I. Introduction 74. II. Application 75. III. Methods 76. IV. Critical Aspects of Technique 77. V. Alternative Methods for Sample Preparation 78. Chapter 13. Flow Cytometry and Sorting of Plant Protoplasts and Cells 79. I. Update 80. II. Introduction 81. III. Application 82. IV. Materials 83. V. Procedures 84. VI. Critical Aspects of the Procedures 85. VII. Controls and Standards 86. VIII. Instruments 87. IX. Results 88. Chapter 14. DNA Content Histogram and Cell-Cycle Analysis 89. I. Introduction 90. II. DNA Content Histogram Basic Principles 91. III. Cell-Cycle Analysis of DNA Content Histograms 92. IV. Critical Aspects of DNA Content and Cell-Cycle Analysis 93. V. Interpretation of DNA Content and Cell-Cycle Histograms 94. VI. Conclusion 95. Chapter 15. Simultaneous Analysis of Cellular RNA and DNA Content 96. I. Update 97. II. Introduction 98. III. Applications 99. IV. Materials 100. V. Staining Procedures Employing AO 101. VI. Staining RNA and DNA with PY and Hoechst 33342 102. VII. Critical Aspects of the Procedures 103. VIII. Controls and Standards 104. IX. Instruments 105. X. Results 106. XI. Comparison of the Methods 107. Chapter 16. Immunochemical Quantitation of Bromodeoxyuridine 108. I. Update 109. II. Introduction 110. III. Applications 111. IV. Materials 112. V. Procedures 113. VI. Critical Aspects of the Procedure 114. VII. Controls and Standards 115. VIII. Instruments 116. IX. Results 117. X. Summary 118. Chapter 17. Cell-Cycle Kinetics Estimated by Analysis of Bromodeoxyuridine Incorporation 119. I. Introduction 120. II. Applications 121. III. Materials 122. IV. Methods 123. V. Critical Aspects of the Procedure 124. Chapter 18. Flow Cytometric Analysis of Cell Division History Using Dilution of Carboxyfluorescein Diacetate Succinimidyl Ester, a Stably Integrated Fluorescent Probe 125. I. Introduction and Background 126. II. Reagents and Solutions 127. III. Preparation and Labeling of Cells 128. IV. Gathering of Information Concurrent with Division 129. V. Analysis of Data 130. VI. Application of Carboxyfluorescein Diacetate Succinimidyl Ester to In Vitro Culture of Lymphocytes 131. VII. Monitoring Lymphocyte Responses In Vivo 132. VIII. Antigen Receptor Transgenic Models 133. Chapter 19. Antibodies Against the Ki-67 Protein 134. I. Introduction 135. II. Application 136. III. Materials and Methods 137. IV. Critical Aspects 138. V. Controls and Standards 139. VI. Examples of Results 140. Chapter 20. Detection of DNA Damage in Individual Cells by Analysis of Histone H2AX Phosphorylation 141. I. Update 142. II. Introduction 143. III. Considerations in the Use of γH2AX as a Measure ofDouble-Stranded Breaks 144. IV. Methods of Analysis 145. V. Typical Results 146. VI. Possible Applications 147. VII. Conclusion 148. Chapter 21. Assays of Cell Viability 149. I. Update 150. II. Introduction 151. III. Changes in Light Scatter During Cell Death 152. IV. Cell Sensitivity to Trypsin and DNase 153. V. Fluorescein Diacetate (FDA) Hydrolysis and PI Exclusion 154. VI. Rh123 Uptake and PI Exclusion 155. VII. PI Exclusion Followed by Counterstaining with Hoechst 33342 156. VIII. Hoechst 33342 Active Uptake and PI Exclusion 157. IX. Controlled Extraction of Low MW DNA from Apoptotic Cells 158. X. Sensitivity of DNA In Situ to Denaturation 159. XI. Detection of DNA Strand Breaks in Apoptotic Cells 160. XII. A Selective Procedure for DNA Extraction from Apoptotic Cells Applicable to Gel Electrophoresis and Flow Cytometry 161. XIII. Comparison of the Methods: Confirmation of the Apoptotic Mode of Cell Death 162. Chapter 22. Difficulties and Pitfalls in Analysis of Apoptosis 163. I. Update 164. II. Introduction 165. III. AI may not be Correlated with Incidence of Cell Death 166. IV. Difficulties in Estimating Frequency of Apoptosis by Analysis of DNA Fragmentation 167. V. The Lack of Evidence is not Evidence for the Lack of Apoptosis 168. VI. Misclassification of Apoptotic Bodies or Nuclear Fragments as Single Apoptotic Cells 169. VII. Apoptosis Versus Necrosis Versus “Necrotic Stage” of Apoptosis 170. VIII. Selective Loss of Apoptotic Cells During Sample Preparation 171. IX. Live Cells Engulfing Apoptotic Bodies Masquerade as Apoptotic Cells 172. X. The Problems with Commercial Kits and Reagents 173. XI. Cell Morphology is still the Gold Standard for Identification of Apoptotic Cells 174. XII. Laser Scanning Cytometry: Have your Cake and Eat it too 175. Chapter 23. Cell Preparation for the Identification of Leukocytes 176. I. Introduction 177. II. Antibodies 178. III. Tandem Fluorochromes 179. IV. Cell Preparation and Staining Procedures 180. V. Titering Antibodies 181. VI. Solutions and Reagents 182. Chapter 24. Multicolor Immunophenotyping 183. I. Update 184. II. Introduction 185. III. Methodology 186. IV. Normal Immunophenotypic Patterns of Maturation 187. V. Abnormal Immunophenotypic Patterns of Maturation 188. Chapter 25. Differential Diagnosis of T-Cell Lymphoproliferative Disorders by Flow Cytometry Immunophenotyping. Correlation with Morphology 189. I. Introduction 190. II. Materials 191. III. Methods 192. IV. Identification of Abnormal T-Cell Population by Flow Cytometry 193. V. Precursor T-Lymphoblastic Lymphoma/Leukemia (T-ALL) 194. VI. Peripheral (Mature/Postthymic) Lymphoma Versus T-ALL 195. VII. Thymocytes from Thymic Hyperplasia/Thymoma Versus T-ALL 196. VIII. Mature T-Cell Lymphoproliferative Disorders 197. IX. Conclusions 198. Chapter 26. B Cell Immunophenotyping 199. I. Update 200. II. Introduction 201. III. Immunophenotyping of B Cell Developmental Stages in the Bone Marrow 202. IV. Peripheral B Cell Populations 203. V. Summary 204. VI. Antigen-Induced B Cell Subsets 205. VII. Antigen-Specific B Cells 206. Chapter 27. Telomere Length Measurements Using Fluorescence In Situ Hybridization and Flow Cytometry 207. I. Introduction 208. II. Background 209. III. Methods 210. IV. Results 211. V. Critical Aspects of Methodology 212. VI. Pitfalls and Misinterpretation of Data 213. VII. Comparison with Other Methods 214. VIII. Applications 215. IX. Future Directions 216. Chapter 28. Sperm Chromatin Structure Assay 217. I. Update 218. II. Introduction 219. III. Applications of the SCSA 220. IV. Materials 221. V. Cell Preparation 222. VI. Cell Staining and Measurement 223. VII. Instruments 224. VIII. Results and Discussion 225. IX. Critical Points 226. Chapter 29. Cell Membrane Potential Analysis 227. I. Introduction 228. II. Materials and Methods 229. III. Improving Cytometry of Membrane Potentials 230. Chapter 30. Measurement of Intracellular pH 231. I. Introduction 232. II. Application 233. III. Materials 234. IV. Cell Preparation and Staining: BCECF or SNARF 235. V. Instruments 236. VI. Critical Aspects 237. VII. Results and Discussion 238. Chapter 31. Intracellular Ionized Calcium 239. I. Introduction 240. II. Flow Cytometric Assay with Indo-1 241. III. Use of Flow Cytometry and Fluo-3 to Measure [Ca2+]i 242. IV. Use of Flow Cytometry and Fluo-3/Fura-Red for Ratiometric Analysis of [Ca2+]i 243. V. Cell Conjugate Assays Combined with Calcium Analysis and Flow Cytometry 244. VI. Pitfalls and Critical Aspects 245. VII. Limitations 246. VIII. Results 247. Chapter 32. Oxidative Product Formation Analysis by Flow Cytometry 248. I. Introduction 249. II. Oxidative Burst 250. III. Phagocytosis 251. IV. Controls and Standards 252. Chapter 33. Phagocyte Function 253. I. Update 254. II. Introduction 255. III. Background 256. IV. Methods 257. V. Results 258. VI. Pitfalls and Misinterpretation of the Data 259. VII. Comparison with Other Methods 260. VIII. Applications and Biomedical Information 261. IX. Future Directions 262. Chapter 34. Analysis of RNA Synthesis by Cytometry 263. I. Update 264. II. Introduction 265. III. Background 266. IV. Methods for Analysis of 5′-Bromouridine Incorporation and DNA Content 267. V. Results of Labeling RNA with 5′-Bromouridine 268. VI. Applications 269. Chapter 35. Analysis of Mitochondria by Flow Cytometry 270. I. Introduction 271. II. Materials and Methods 272. III. Critical Aspects 273. Chapter 36. Analysis of Platelets by Flow Cytometry 274. I. Introduction 275. II. Applications 276. III. Materials 277. IV. Cell Preparation 278. V. Staining 279. VI. Critical Aspects 280. VII. Standards 281. VIII. Instrument 282. IX. Results and Discussion 283. Chapter 37. Detection of Specific Microorganisms in Environmental Samples Using Flow Cytometry 284. I. Introduction 285. II. Preparation of Water Samples for Flow Cytometric Analysis 286. III. Staining of Organisms from Water Samples for Flow Cytometric Analysis 287. IV. Flow Cytometric Analysis of Water Samples 288. V. Instrumentation Developments for Environmental Applications 289. VI. The Future of Flow Cytometry within Environmental Microbiology 290. Chapter 38. Flow Cytometric Analysis of Microorganisms 291. I. Introduction 292. II. Experimental Approaches 293. III. Applications in Medical and Food Microbiology 294. IV. Conclusion 295. Chapter 39. Flow Cytometry in Malaria Detection 296. I. Introduction 297. II. Applications 298. III. Materials 299. IV. Cell Preparation and Staining 300. V. Critical Aspects of the Preparation and Staining Procedures 301. VI. Standards 302. VII. Instruments 303. VIII. Results and Discussion 304. IX. Comparison of Methods 305. Index