Practical Cell Analysis -- Contents -- Preface -- Acknowledgments -- 1 Getting Started (and Getting the Cells) -- 1.1 INTRODUCTION -- 1.2 THE DRIVING NEED -- 1.3 PRIMARY AND CULTURED CELLS -- 1.4 CHOOSING A CULTURED CELL -- 1.5 CHOOSING PRIMARY CELLS -- 1.6 EASILY OBTAINABLE PRIMARY CELLS -- 1.7 PRIMARY CELLS FROM TISSUES -- 1.8 PURIFYING PRIMARY CELLS -- 1.9 HOW LONG DO PRIMARY CELLS REMAIN PRIMARY? -- 1.10 OBTAINING PRIMARY CELLS FROM A COMMERCIAL SOURCE -- 1.11 BACTERIA AND YEAST -- 1.12 PRACTICAL ASPECTS OF CELL CULTURE -- 1.13 SAFETY ASPECTS OF PRIMARY AND TRANSFORMED CELL LINES -- 1.14 TRANSFECTION OF PRIMARY AND TRANSFORMED CELL LINES -- 1.15 CONCLUSION -- REFERENCES -- 2 The Cell-Culture Laboratory (Tools of the Trade) -- 2.1 INTRODUCTION -- 2.2 ISSUES CONCERNING A CELL LABORATORY -- 2.3 SETTING UP A CELL CULTURE LABORATORY -- 2.4 CELL LINE STORAGE -- 2.5 PERSONAL PROTECTIVE EQUIPMENT -- 2.6 CELL AND SAMPLE HANDLING -- 2.7 COMMON ANALYTICAL INSTRUMENTATION FOR CELL CULTURE -- 2.8 CONSIDERATIONS WHEN SETTING UP A CELL-CULTURE LABORATORY -- 2.9 ESTABLISHING AND REGULATING A CULTURE FACILITY -- 2.10 CONCLUSION -- REFERENCES -- 3 Maintaining Cultures -- 3.1 INTRODUCTION -- 3.2 MEDIUM -- 3.3 THE USE OF MEDIUM IN ANALYSIS, AND ALTERNATIVES -- 3.4 CULTURING CELLS -- 3.5 GROWING CELLS IN THREE DIMENSIONS -- 3.6 STERILITY AND CONTAMINATION OF CULTURE -- 3.7 STORAGE OF CELL SAMPLES AND CELL LINES -- PROTOCOL 3.2: CRYOPRESERVATION OF MAMMALIAN CELLS -- PROTOCOL 3.3: RETRIEVAL OF CELLS FROM LIQUID-NITROGEN STORAGE -- 3.8 CONCLUSION -- REFERENCES -- 4 Microscopy of Cells -- 4.1 INTRODUCTION -- 4.2 MICROSCOPE TYPES -- 4.3 CULTURING CELLS FOR MICROSCOPY -- 4.4 SIGNALS, BACKGROUND, AND ARTIFACTS IN OPTICAL MICROSCOPY -- 4.5 STAINING CELLS FOR FLUORESCENCE MICROSCOPY -- PROTOCOL 4.1 FIXATION OF CELLS FOR IMMUNOCHEMICAL STAINING -- 4.6 MULTIPLE LABELS.

4.7 VIABILITY AND TWO-PHOTON MICROSCOPY -- 4.8 SPATIAL RESOLUTION IN OPTICAL MICROSCOPY -- 4.9 IMAGE SATURATION AND INTENSITY -- 4.10 ATOMIC FORCE AND ENVIRONMENTAL SCANNING ELECTRON MICROSCOPY -- 4.11 CONCLUSION -- REFERENCES -- 5 Separating Cells -- 5.1 INTRODUCTION -- 5.2 THE CELL SAMPLE -- 5.3 LABEL-FREE (INTRINSIC) SEPARATIONS -- 5.4 IMMUNOMAGNETIC SORTING -- 5.5 CELL-AFFINITY CHROMATOGRAPHY -- 5.6 AFFINITY CHEMISTRY CONSIDERATIONS IN CAC AND MACS SEPARATIONS -- PROTOCOL 5.1: SCREENING OF ANTIBODY CLONES -- 5.7 ELUTION IN CELL-AFFINITY CHROMATOGRAPHY -- 5.8 NONSPECIFIC BINDING IN CELL SEPARATIONS -- 5.9 SEPARATION OF RARE CELLS -- 5.10 FLUORESCENCE-ACTIVATED CELL SORTING -- 5.11 SORTING PARAMETERS -- 5.12 OTHER SEPARATION TECHNIQUES AND CONSIDERATIONS -- 5.13 CONCLUSION -- REFERENCES -- 6 Flow Cytometry: Cell Analysis in the Fast Lane -- 6.1 INTRODUCTION -- 6.2 THE CELL SAMPLE -- 6.3 FLOW CYTOMETER FUNCTION -- 6.4 OBTAINING OR FINDING A FLOW CYTOMETER -- 6.5 USING FLOW CYTOMETERS -- 6.6 SETTING UP A FLOW CYTOMETER FOR MULTI-COLOR STAINING -- 6.7 ANALYZING FLOW CYTOMETRY DATA -- 6.8 EXAMPLE FLOW-CYTOMETRY ASSAYS -- 6.9 NO-FLOW CYTOMETRY -- 6.10 CONCLUSION -- REFERENCES -- 7 Analyzing Cells with Microfluidic Devices -- 7.1 INTRODUCTION -- 7.2 ADVANTAGES OF MICROFLUIDICS -- 7.3 CONSIDERATIONS OF MICROFLUIDICS AND CELLS -- 7.4 OBTAINING MICROFLUIDIC CELL DEVICES -- 7.5 MICROFLUIDIC FLOW CYTOMETRY -- 7.6 CELL SEPARATIONS -- 7.7 ANALYSIS OF CELL PRODUCTS -- 7.8 CELL CULTURE -- PROTOCOL 7.1: LOW-SHEAR CELL-CULTURE CHIP -- 7.9 CONCLUSION -- REFERENCES -- 8 Statistical Considerations -- 8.1 INTRODUCTION -- 8.2 TYPES OF ERROR -- 8.3 FIGURES OF MERIT IN STATISTICAL ANALYSIS OF CELLS -- 8.4 LIMITS OF DETECTION AND QUANTITATION (OF CELLS) -- 8.5 METHODS TO IMPROVE CELL STATISTICS -- 8.6 COMPARING ANALYTICAL VALUES -- 8.7 REJECTING DATA: PROCEED WITH CAUTION.

8.8 CONCLUSION -- REFERENCES -- 9 Protocols, Probes, and Standards -- 9.1 INTRODUCTION -- 9.2 CELL TRANSFECTION AND IMMORTALIZATION (CHAPTER 1) -- PROTOCOL 9.1: TRANSFECTING CELLS WITH POLYAMINE REAGENTS -- PROTOCOL 9.2: STABLE TRANSFECTION USING POLYAMINE DELIVERY -- PROTOCOL 9.3: TRANSFECTION USING ELECTROPORATION -- PROTOCOL 9.4: CELL IMMORTALIZATION USING hTERT TRANSFECTION -- 9.3 CALCULATING RELATIVE CENTRIFUGAL FORCE (RCF) AND CENTRIFUGE ROTOR SPEED (CHAPTER 2) -- 9.4 FLUORESCENCE METHODS (CHAPTERS 4 AND 6) -- PROTOCOL 9.4: APOPTOSIS DETECTION USING FLUOROPHORE-CONJUGATED ANNEXIN-V AND A VIABILITY DYE -- PROTOCOL 9.5: APOPTOSIS DETECTION USING FLUOROGENIC CASPASE PROBES -- 9.5 SURFACE MODIFICATIONS FOR CELL ANALYSIS (CHAPTERS 5 AND 7) -- PROTOCOL 9.6: COVALENT LINKAGE OF PROTEINS (NONANTIBODY) TO GLASS BY MICROCONTACT IMPRINTING -- PROTOCOL 9.7: COVALENT LINKAGE OF ANTIBODIES TO GLASS -- PROTOCOL 9.8: NONCOVALENT ATTACHMENT OF ANTIBODIES TO GLASS #1 -- PROTOCOL 9.9: NONCOVALENT ATTACHMENT OF ANTIBODIES TO GLASS OR PDMS #2 -- PROTOCOL 9.10: BLOCKING ENDOGENOUS BIOTIN -- 9.6 FLOW CYTOMETRY AND CELL SEPARATIONS (CHAPTERS 5 AND 6) -- PROTOCOL 9.11: CELL CYCLE MEASUREMENTS BY FLOW CYTOMETRY -- PROTOCOL 9.12: ANTIGEN DENSITY MEASUREMENTS IN FLOW CYTOMETRY -- PROTOCOL 9.13: ANTIGEN DENSITY MEASUREMENTS USING FLUORESCENCE CORRELATION SPECTROSCOPY -- PROTOCOL 9.14: CELL PROLIFERATION USING ANTI-CD71 STAINING (CHAPTERS 4 AND 6) -- 9.7 FLUORESCENT LABELS AND FLUOROGENIC PROBES (CHAPTERS 4-7) -- REFERENCES -- Index.