Tissue Culture Techniques: An Introduction

ISBN-13: 9780817636432 / Angielski / Miękka / 1994 / 260 str.

B. Martin; Bernice M. Martin; Bernice Martin

Opis

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl II INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I STERILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Aseptic Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Physical manipulations Use of the sterile cabinet (hood) Sterilization Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Heat Radiation Toxic gas Filtration Antibiotics Quality Control of Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Routine labeling Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 vi CONTENTS ROUTINE CELL CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Feeding Schedules and Media Components . . . . . . . . . . . . . . . . . . . . 29 General properties of media and salt solutions water as a reagent. Establishingfeeding schedules Subcultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Solutions and methods for adherent cells Common enzyme solutions Inoculating (seeding) the cultures Cell Enumeration and Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Hemocytometer Particle counter Cell viability Putting Routine Methods to Work . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Normal cell growth characteristics Detecting and Disposing of Contamination . . . . . . . . . . . . . . . . . . . . 66 Bacteria and fUngi Fungi Mycoplasma Viruses Dealing with contamination Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Inadequate cell growth Recurrent contamination When to call your vendor Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Biological hazards Chemical hazards Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 EXPERIMENTS IN CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . 91 II Alterations of the Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Serum Treatments of serum Plasma-derived serum Serum-free and low-protein media Substrata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Coatingplasticware with solutions Alterations with polymers Using cells to coat the plasticware Culturing cells on microcarriers Altering the Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Temperature changes Gaseous changes Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 CONTENTS vii PRIMARY CELL CULTURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Dissection Enzymatic dissociation methods Nonenzymatic isolation Purification of cell suspensions Consideringyield and survival Chatacterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ."

Spis treści

1 Introduction.- 2 Sterility.- Aseptic Technique.- Physical manipulations • Use of the sterile cabinet (hood).- Sterilization Methods.- Heat • Radiation • Toxic gas • Filtration •Antibiotics.- Quality Control of Sterilization.- Routine labeling.- Suggested Readings.- Exercises.- 3 Routine Cell Culture.- Feeding Schedules and Media Components.- General properties of media and salt solutions • Water as a reagent • Establishing feeding schedules.- Subcultivation.- Solutions and methods for adherent cells • Common enzyme solutions • Inoculating (seeding) the cultures.- Cell Enumeration and Cell Viability.- Hemocytometer • Particle counter • Cell viability.- Putting Routine Methods to Work.- Normal cell growth characteristics.- Detecting and Disposing of Contamination.- Bacteria and fungi • Fungi • Mycoplasma • Viruses • Dealing with contamination.- Troubleshooting.- Inadequate cell growth • Recurrent contamination • When to call your vendor.- Safety.- Biological hazards • Chemical hazards.- Suggested Readings.- Problem Set.- Exercises.- 4 Experiments in Culture.- Alterations of the Media.- Serum • Treatments of serum • Plasma-derived serum • Serum-free and low-protein media.- Substrata.- Coating plasticware with solutions •Alterations with polymers •Using cells to coat the plasticware • Culturing cells on microcarriers.- Altering the Environment.- Temperature changes • Gaseous changes.- Problem Set.- Exercises.- 5 Primary Cell Culture.- Isolation.- Dissection • Enzymatic dissociation methods • Nonenzymatic isolation • Purification of cell suspensions • Considering yield and survival.- Characterization.- Preparation • Fixation andpermeahilization • Identification.- Exercise.- 6 Cell Preservation.- Freezing the Cells.- Thawing the Cells.- Equipment • Solutions.- Problem Set.- 7 Cell Cloning.- Isolation.- Limiting dilution • Cloning cylinders.- Assisting Clonal Survival.- Using conditioned medium • Using feeder layers.- Problem Set.- 8 Culture Changes.- The Loss of Differentiated Phenotype.- Immortality and Aging.- Cell Transformation.- Chemical transformation • Viral transformation • Transformed cells from tumors.- Transfection (Gene Transfer).- Methods • Evaluation.- Suggested Readings.- Problem Set.- Exercises.- 9 Information for New Cell Studies.- Searching in the Literature.- Using Advertising and Catalogs.- References, Bibliographies, and Databases.- Appendices.- A The Cell Cycle.- Suggested Readings.- B Media and Salt Solutions.- Media.- Salt Solutions.- C Vendors.- D Glossary.- E Answers to Problem Sets and Exercises.