Cell Culture Basics Handbook - Thermo Fisher Scientific

1 Cell Culture Basics Handbook Includes transfection Gibco Education Culture your career Information in this document is subject to change without notice. DISCLAIMER. Thermo Fisher Scientific AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS. DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR. A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE. TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY. STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL. DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE. USE THEREOF. 2016 Thermo Fisher Scientific Inc. All rights reserved. COL31122 0516. Contents 1. Introduction.

2 1. Purpose of the Handbook .. 1. Introduction to Cell Culture .. 2. What is cell Culture ? .. 2. Finite vs. continuous cell line .. 2. Culture conditions .. 2. Cryopreservation .. 2. Morphology of cells in Culture .. 3. Applications of cell Culture .. 3. 2. Cell Culture Laboratory .. 4. Safety .. 4. Biosafety levels .. 4. SDS .. 5. Safety equipment .. 5. Personal protective equipment (PPE) .. 5. Safe laboratory practices .. 5. Cell Culture Equipment .. 6. Basic equipment .. 6. Expanded equipment .. 6. Additional supplies .. 6. Cell Culture Laboratory .. 7. Aseptic work area .. 7. Cell Culture hood .. 7. Cell Culture hood layout .. 8. Incubator .. 9. Storage .. 9. Cryogenic storage .. 10. Cell counter .. 10. Aseptic Technique .. 11. Introduction .. 11. Sterile work area .. 11. Good personal hygiene .. 11. Sterile reagents and media .. 12. Sterile handling.

3 12. Cell Culture Basics | iii For educational purposes only. Aseptic Technique Checklist .. 13. Biological Contamination .. 14. Introduction .. 14. Bacteria .. 14. Yeasts .. 15. Molds .. 15. Viruses .. 16. Mycoplasma .. 16. Cross-contamination .. 17. Using antibiotics .. 17. 3. Cell Culture Basics .. 18. Cell Lines .. 18. Selecting the appropriate cell line .. 18. Acquiring cell lines .. 18. Culture Environment .. 19. Adherent vs. suspension Culture .. 19. Media .. 20. pH .. 21. CO2 .. 21. Temperature .. 21. Cell Morphology .. 22. Mammalian cells .. 22. Variations in mammalian cell morphology .. 22. Morphology of 293 cells .. 23. Insect cells .. 24. Morphology of Sf21 cells .. 24. Morphology of Sf9 cells .. 25. 4. Cell Culture Methods .. 26. Guidelines for Maintaining Cultured cells .. 26. What is subculture? .. 26. When to subculture? .. 27.

4 Media recommendations for common cell lines .. 28. iv | Cell Culture Basics For educational purposes only. Dissociating adherent cells .. 30. TrypLE dissociation enzymes .. 30. Subculturing Adherent cells .. 31. Materials needed .. 31. Protocol for passaging adherent cells .. 31. Notes on subculturing adherent insect cells .. 32. Subculturing Suspension cells .. 33. Passaging suspension cultures .. 33. Suspension Culture vessels .. 33. Materials needed .. 34. Protocol for passaging suspension cells .. 34. Notes on subculturing suspension insect cells .. 36. Freezing cells .. 37. Cryopreservation .. 37. Guidelines for cryopreservation .. 37. Freezing medium .. 38. Materials needed .. 38. Cryopreserving cultured cells .. 39. Thawing Frozen cells .. 40. Guidelines for thawing .. 40. Materials needed .. 40. Thawing frozen cells .. 40. 5. Transfection Basics .

5 41. Introduction to Transfection .. 41. What is transfection? .. 41. Terminology .. 41. Applications .. 42. Types of Transfection .. 43. Transient transfection .. 43. Stable transfection .. 43. Choosing a transfection strategy .. 44. Gene Delivery Technologies .. 46. Cationic lipid mediated delivery .. 48. v For educational purposes only. Calcium phosphate coprecipitation .. 49. DEAE-dextran mediated delivery .. 50. Delivery by other cationic polymers .. 51. Viral delivery .. 52. Electroporation .. 53. Other physical delivery methods .. 54. Cationic Lipid Mediated Transfection .. 55. Mechanism .. 55. Cationic lipid transfection reagents .. 56. Virus Mediated Gene Transfer .. 58. Key properties of viral vectors .. 58. Common viral vectors .. 58. Neon Transfection System .. 60. Selection of Stable Transfectants .. 62. Selection antibiotics for eukaryotic cells .

6 62. Reporter Gene Assays .. 63. Transfection assays .. 63. Gene regulation assays .. 64. Common reporter genes .. 64. RNAi and Noncoding RNA Research .. 65. Glossary of common RNAi terms .. 65. How RNAi works .. 66. siRNA analysis .. 66. miRNA analysis .. 67. Choosing an RNAi approach .. 68. 6. Transfection Methods .. 69. Factors Influencing Transfection Efficiency .. 69. Cell type .. 69. Cell health and viability .. 70. Confluency .. 71. Media .. 71. Serum .. 71. Antibiotics .. 72. vi | Cell Culture Basics For educational purposes only. Type of molecule transfected .. 72. Transfection method .. 72. Selecting a Transfection Method (nonviral) .. 73. Continuous cell lines .. 73. Primary cells and finite cultures .. 74. Selecting a Viral DNA Delivery System .. 75. Expression in mammalian cells .. 75. Expression in insect cells .. 76. Guidelines for Plasmid DNA Transfection.

7 77. Vector considerations .. 77. Quality of plasmid DNA .. 77. Gene product and promoter .. 78. Controls .. 78. Optimization of Plasmid DNA Transfection .. 78. Considerations for calcium phosphate coprecipitation .. 79. Considerations for cationic lipid mediated delivery .. 80. Considerations for electroporation .. 82. Selection of Stable Transfectants .. 83. Before starting .. 83. Kill curve .. 83. Selection workflow .. 83. Selecting an RNAi Strategy .. 85. siRNA vs. vector approaches .. 85. Nonvector siRNA technologies .. 86. siRNA transfection .. 88. Vector-mediated RNAi .. 89. Guidelines for RNA Transfection .. 90. RNAi workflow .. 90. Handling RNA .. 91. Transfection efficiency .. 91. Positive controls .. 91. Negative controls .. 91. Cotransfection .. 92. siRNA quality .. 92. vii For educational purposes only. siRNA quantity .. 93. Volume of transfection reagent.

8 93. Cell density .. 93. Exposure to transfection agent/siRNA complexes .. 93. Presence of serum during transfection .. 94. Tips for a successful siRNA experiment .. 94. Optimization of siRNA Transfection .. 95. Factors affecting siRNA transfection efficiency .. 95. Appendix .. 96. Troubleshooting .. 96. Cell Culture and Transfection Products .. 97. Cell lines .. 97. Media for mammalian cell Culture .. 98. Media for insect cell Culture .. 99. Serum products for cell Culture .. 99. Laboratory reagents for cell Culture .. 100. Antibiotics and antimycotics .. 101. Growth factors and purified proteins .. 101. Accessory products for cell Culture .. 102. Transfection reagents .. 102. Neon Transfection System .. 103. RNA interference .. 103. Additional Resources .. 104. Mammalian and insect cell cultures .. 104. Cell and tissue analysis .. 104. Transfection selection tool.

9 104. Safety data sheets .. 104. Certificate of analysis .. 104. Technical support .. 104. References .. 105. Notes .. 108. viii | Cell Culture Basics For educational purposes only. 1. Introduction Purpose of the Handbook The Cell Culture Basics Companion Handbook is a supplement to the Cell Culture Basics instructional videos available online at The Handbook and videos are intended as an introduction to cell Culture Basics . The first four chapters of the Handbook focus on cell Culture , covering topics such as getting familiar with the requirements of a laboratory dedicated to cell Culture experiments, laboratory safety, aseptic technique, and microbial contamination of cell cultures, as well as providing basic methods for passaging, freezing, and thawing cultured cells . The subsequent two chapters of the Handbook focus on various transfection technologies and provide general guidelines for the selection of the appropriate transfection method, the transfection of cells with plasmid DNA, oligonucleotides, and RNA, as well as Culture preparation for in vitro and in vivo transfection and selection of the transfected cells .

10 The information and guidelines presented in the Handbook and the instructional videos focus on cell lines (finite or continuous) and omit experiments and techniques concerning primary cultures and stem cells , such as isolating and disaggregating tissues, reprogramming cells into pluripotent stem cells , or differentiating stem cells into various lineages. Note that while the Basics of cell Culture experiments share certain similarities, cell Culture conditions vary widely for each cell type. Deviating from the Culture conditions required for a particular cell type can result in different phenotypes being expressed; we therefore recommend that you familiarize yourself with your cell line of interest, and closely follow the instructions provided with each product you are using in your experiments. Cell Culture Basics | 1. For educational purposes only.