Leucocytes: Methods and Protocols

Cover_978-1-61779-526-8......Page 1
FrontMatter......Page 2
Leucocytes......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 10
1. Introduction......Page 14
2.1. Mutagenesis Protocols......Page 15
2.3. FACS Analysis......Page 16
3.1. Quantification of Mouse Peripheral Blood Leukocytes Using a Haematology Analyser......Page 17
3.1.2. Blood Cell Count......Page 18
3.2.3. Blocking and Staining......Page 20
3.3. Bone Marrow Cell Extraction......Page 21
3.5. Multiplex Analysis of Cytokine by Luminex......Page 22
4. Notes......Page 24
References......Page 26
1. Introduction......Page 28
2.1. Standard and Sequential ELISA......Page 29
2.2. Multiplex Materials and Equipment......Page 30
3.1. Optimizing Antibody Concentrations (the Checkerboard)......Page 31
3.2. Determining the Optimal Blocking and Dilution Buffer for the Sample Matrix (the Dose–Response)......Page 33
3.3. Determining an Analyte’s Concentration in a Given Sample (the Standard ELISA)......Page 34
3.4. The Sequential ELISA......Page 35
3.5. The Multiplex ELISA......Page 36
3.5.1. General Method......Page 38
3.5.3. Cost Comparison......Page 39
4. Notes......Page 40
References......Page 43
1. Introduction......Page 44
2.4. Flow Cytometry Staining......Page 46
3.3. Cell Preparation......Page 48
3.5.1. Flow Cytometry Analysis of Cell Cycling with Ki67 and Hoechst33342 Stains on BM Cells......Page 49
3.5.2. Flow Cytometry Analysis of Cell Proliferation by BrdU Stain......Page 52
4. Notes......Page 54
References......Page 56
1. Introduction......Page 58
2.2. Tissue Harvest......Page 60
2.4. Phenotypic Stains......Page 61
3.1. Animal Treatment and Tissue Harvest......Page 62
3.2.1. Blood......Page 64
3.3.1. Single-Color Control on Blood Leukocytes and Adjustment of the Flow Cytometer......Page 65
3.3.2. Fully Perfused Blood Leukocyte-Positive Controls......Page 66
3.3.3. Hematopoietic Stem Cell Staining......Page 67
3.3.4. Myeloid Progenitors Staining......Page 68
3.3.5. Endothelial Cells, MSCs, and Osteoblasts......Page 70
4. Notes......Page 73
References......Page 75
1. Introduction......Page 78
2.3.1. FLICA Poly Caspases Assay Kit......Page 81
2.8. Confocal Microscopy......Page 82
3.2. Depolarization of the Mitochondrial Transmembrane Potential......Page 83
3.3. Activation of Caspases......Page 85
3.3.2. Caspase-Glo 3/7 Assay......Page 86
3.4. Extracellular Exposure of Phosphatidylserine, Altered Light Scatter, Plasma Membrane Permeabilization, and Secondary Necrosis......Page 87
3.4.1. Annexin V and 7-AAD Staining......Page 88
3.4.2. Amine-Reactive Dyes to Distinguish Permeable Membranes......Page 89
3.6. Morphology......Page 90
3.8. Clearance of Apoptotic Neutrophils by MDMs......Page 91
3.8.3. MDM Phagocytosis of Apoptotic Neutrophils......Page 92
4. Notes......Page 93
References......Page 97
1. Introduction......Page 100
3. Positive and Negative Regulation of Neutrophil Production......Page 101
4. Multifunctional Roles for Neutrophil Granule Proteins and NADPH Oxidase......Page 102
5. Neutrophil Extracellular Traps......Page 104
6. Retrograde Neutrophil Chemotaxis and Antigen Presentation......Page 105
7. How to Subdivide the Neutrophil Population?......Page 106
8. Understanding Neutrophil Persistence During Inflammation......Page 107
References......Page 109
1. Introduction......Page 114
2. Materials......Page 115
3.2. A Density Gradient Method Using Percoll for Isolation of Neutrophils from Human Peripheral Blood......Page 117
3.3. Using a Cell Preparation Tube to Isolate Neutrophils from Human Peripheral Blood ( see Note 8)......Page 118
3.5. Isolation of Neutrophils from Human Saliva......Page 120
3.6. Using Negative Immunomagnetic Separation to Isolate Murine Blood Neutrophils......Page 121
3.7. Isolation of Neutrophils from Mouse Bone Marrow......Page 122
3.9. Isolation of Neutrophils from Peritoneal Exudate......Page 123
4. Notes......Page 124
References......Page 125
1. Introduction......Page 128
2.1. Human PMN Isolation ( see Note 1)......Page 129
3. Methods......Page 130
3.1. Human PMN Isolation......Page 132
3.4. Measurement of Oxidative Burst Using Dihydrorhodamine 123 and Flow Cytometry......Page 133
3.6. Measurement of PMN Oxidative Burst in Mouse Whole Blood......Page 134
4. Notes......Page 135
References......Page 136
1. Introduction......Page 138
2.1. Synthesis of Fluorogenic Substrates......Page 141
2.3. Purification of Blood Neutrophils......Page 142
3. Methods......Page 143
3.1.2. Synthesis of EDDnp......Page 144
3.1.5. Coupling Fmoc-Glu( γ-OH)-EDDnp to Resin\r......Page 145
3.3. Determination of the Concentrations of Purified Substrates......Page 146
3.5. Measuring Membrane-Bound Elastase, Proteinase 3, and Cathepsin G Activity......Page 147
4. Notes......Page 150
References......Page 151
1. Introduction......Page 152
2.1. Murine Classification of Blood Monocytes......Page 153
2.2. Human Classification of Blood Monocytes......Page 155
2.3. Conservation Across Species......Page 156
2.4. Origins and Fates of Mouse Monocytes......Page 157
3.1. Classically and Alternatively Activated Macrophages......Page 159
3.2. Cross-species Conservation of Macrophages Activation Spectrums......Page 160
3.4. Deriving Macrophages for Immunological Studies......Page 161
4.1. Sensing......Page 162
4.2. Chemotaxis......Page 163
4.4. Adaptive Stimulation......Page 164
References......Page 165
1. Introduction......Page 170
2.1. Transgenic Mice Generation......Page 171
2.2. EGFP Expression Characterization in Embryo and Adult Tissues......Page 172
3.1.1. Transgene Construction and Preparation for Pronuclear Injection......Page 173
3.1.2. Harvesting Fertilized Eggs......Page 175
3.1.3. Pronuclei Injection......Page 176
3.1.5. Genomic DNA Extraction from Tail Tip Biopsies......Page 177
3.1.6. PCR Screening of Positive Founders/TG Litter......Page 178
3.2.2. Whole Embryo Examinations......Page 179
3.2.4. Resident Peritoneal Macrophages......Page 181
3.2.5. Pulmonary Macrophages......Page 182
3.2.7. Culture of Osteoclasts......Page 183
3.2.9. Isolation of Macrophages from Intestinal Lamina Propria......Page 184
3.2.10. Examination of EGFP Expression in Tissue Sections......Page 185
3.2.11. Examination of Langerhans Cell in the Epidermal Sheet of the Skin......Page 186
4. Notes......Page 187
References......Page 188
1. Introduction......Page 190
2. Materials......Page 191
3. Methods......Page 192
4. Notes......Page 193
References......Page 194
1. Introduction......Page 196
3.1. Ficoll-Hypaque Density Centrifugation to Isolate PBMC (from RBC and PMN)......Page 197
3.2. MACS Separation for Positive Selection of CD14 + Monocytes......Page 198
References......Page 199
1. Introduction......Page 202
2. Materials......Page 203
3.1. Infection of Macrophages with Cryptococcus neoformans......Page 204
3.2. Phagocytosis Assay......Page 205
3.3. Cryptococcus neoformans Killing Assay......Page 206
4. Notes......Page 207
References......Page 210
1. Introduction......Page 212
2.2. Stimulation......Page 218
2.3. Measurement......Page 219
3.1. Differentiation of Murine BMDCs ( see Note 1)......Page 221
3.2. Stimulation......Page 222
3.3. Measurement......Page 224
4.1. Differentiation of Murine BMDCs......Page 225
4.2. Stimulation......Page 227
4.3. Measurement......Page 229
4.4.1. Storage of Bone Marrow in Liquid Nitrogen......Page 230
4.4.3. Quantification of Intracellular Pro-IL-1 b by ELISA......Page 231
4.4.4. Protein Precipitation from Supernatants......Page 232
4.4.6. Fluorescent Caspase-1 Substrates......Page 233
References......Page 234
1. Introduction......Page 236
2.2. Quantitative Real-Time PCR......Page 240
2.3. Arginine Transport......Page 241
3.1. Cell Culture ( 12)......Page 242
3.2. Determination of Arginine Transporters by mRNA Expression ( 13)......Page 243
3.4. Arginine Catabolism ( 6, 9) (Fig.  3)......Page 245
4. Notes......Page 247
References......Page 248
1. Introduction......Page 250
2.1. Culture of YT Cells and Purification of Human and Mouse Lymphocytes......Page 251
2.2. Preparation of Cell Lysates and Immunoblotting......Page 253
2.5. Confocal Immunofluorescence......Page 254
3.1. Culture of YT Cells and Purification of Human and Mouse Lymphocytes......Page 255
3.2. Preparation of Cell Lysates and Immunoblotting......Page 256
3.3. Detection of Granzyme B by FACS Analysis......Page 257
3.5. Indirect Immunofluorescence for Confocal Microscopy......Page 258
4. Notes......Page 261
References......Page 263
1. Introduction......Page 264
2.4. Substrates......Page 265
3. Methods......Page 266
3.1.2. IETD-AFC or Abz-IEPDSSMES(K-dnp)......Page 267
3.2. Preparation of Mouse Splenocyte Lysates......Page 268
3.3. Detection of Mouse GrB Activity in Splenocytes......Page 269
3.4. Preparation of Human Primary CD8 + T Cell Lysates......Page 270
3.5. Detection of Human GrB Activity in Primary CD8+ T Cells......Page 271
4. Notes......Page 272
References......Page 273
1. Introduction......Page 274
2.3. Epithelial Cell Defective Models of IBD......Page 275
3.1.1. Animals and Reagents......Page 277
3.1.4. Enrichment for CD4 + T Cells (Miltenyi Kit)......Page 278
3.1.6. Fluorescent Labelling of CD4 + T Cells for Cell Sorting......Page 279
3.1.8. Disease Progression and Analysis......Page 281
3.1.9. Tissue Collection, Sampling, and Scoring for Colitis......Page 283
5. Transfer Colitis in Winnie......Page 285
References......Page 287
1.1. Origin and Significance of Nitrite in Urine......Page 290
1.2.2. Derivatization and GC-MS of Nitrite......Page 293
2. Materials......Page 295
3.1. Derivatization and Extraction Procedures......Page 297
3.2. Quality Control for Urinary Nitrite......Page 298
3.3. GC-MS and Mode of Quantification......Page 299
3.4.1. Contribution of Laboratory Materials and Laboratory Air to Urinary Nitrite......Page 300
Effects of Diuretics and Interference by Carbonate/Bicarbonate......Page 301
4. Notes......Page 302
References......Page 305
BackMatter_INDEX......Page 308