URE T ULC LL E C BASICS HANDBOOK

1 CELL CULTUREBASICS HANDBOOKM aster basic cell culture techniques and achieve consistent resultsMake the connectionInformation in this document is subject to change without 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.

2 2020 Thermo Fisher Scientific Inc. All rights reserved. COL011985 0820 ContentsCell culture BASICS | iiiFor educational purposes Introduction ..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 ..3 Morphology of cells in culture ..3 Applications of cell culture ..32. 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.

3 6 Expanded equipment ..7 Additional supplies ..7 Cell culture laboratory ..7 Aseptic work area ..7 Cell culture hood ..7 Cell culture hood layout ..9 Incubator ..10 Storage ..10 Cryogenic storage ..11 Cell counter ..11 Aseptic technique ..12 Introduction ..12 Sterile work area ..12 Good personal hygiene ..12 Sterile reagents and media ..12 Sterile handling ..13 Aseptic technique checklist ..14iv | Cell culture basicsFor educational purposes contamination ..15 Introduction ..15 Bacteria ..15 Yeasts ..16 Molds ..16 Viruses ..17 Mycoplasmas .. 17 Cross-contamination ..18 Using antibiotics.

4 183. Cell culture BASICS ..19 Cell lines ..19 Selecting the appropriate cell line ..19 Acquiring cell lines ..20 Culture environment ..20 Adherent vs. suspension culture ..20 Media ..21 Serum ..22 Cell culture plastics ..23pH ..25CO2 ..25 Temperature ..25 Cell morphology .. 26 Mammalian cells ..26 Variations in mammalian cell morphology ..26 Morphology of 293 cells ..27 Insect cells ..28 Morphology of Sf21 cells ..28 Morphology of Sf9 cells ..294. Cell culture methods ..30 Guidelines for maintaining cultured cells ..30 What is subculture? ..30 When to subculture ..31 Media recommendations for common cell lines.

5 32 TrypLE dissociation enzymes ..34 Cell culture BASICS | vFor educational purposes adherent cells ..35 Materials needed ..35 Protocol for passaging adherent cells ..35 Notes on subculturing adherent insect cells ..37 Subculturing suspension cells ..38 Passaging suspension cultures ..38 Suspension culture vessels ..38 Passaging suspension cultures ..39 Protocol for passaging suspension cells ..39 Notes on subculturing suspension insect cells ..41 Freezing cells ..42 Cryopreservation ..42 Guidelines for cryopreservation ..42 Freezing medium ..43 Materials needed ..44 Cryopreserving cultured cells.

6 44 Thawing frozen cells ..45 Guidelines for thawing ..45 Materials needed ..46 Thawing frozen cells ..465. Transfection BASICS ..47 Introduction to transfection ..47 What is transfection? ..47 Terminology ..47 Applications ..48 Types of transfection ..49 Transient transfection ..49 Stable transfection ..50 Choosing a transfection strategy ..51 Gene delivery technologies ..54 Cationic lipid mediated delivery ..54 Calcium phosphate coprecipitation ..56 DEAE-dextran mediated delivery ..57 Delivery by other cationic polymers ..58 Viral delivery ..59 Electroporation ..61 Other physical delivery methods.

7 62 Cationic lipid mediated transfection ..63 Mechanism ..63 Cationic lipid transfection reagents ..64 Virus-mediated gene transfer ..66 Key properties of viral vectors ..66 Common viral vectors ..67 Neon transfection system ..69 Selection of cells for stable transfection ..71 Selection antibiotics for eukaryotic cells ..71 Reporter gene assays ..73 Transfection assays ..73 Gene regulation assays ..73 Common reporter genes ..74 RNAi and noncoding RNA research ..75 Glossary of common RNAi terms ..75 How RNAi works ..76siRNA analysis ..76miRNA analysis ..77 Choosing an RNAi approach ..786.

8 Transfection methods ..79 Factors influencing transfection efficiency ..79 Cell type ..79 Cell health and viability ..80 Confluency ..81 Media ..81 Serum ..82 Antibiotics ..82 Type of molecule transfected ..83 Transfection method ..83 Selecting a transfection method (nonviral) ..84 Continuous cell lines ..84 Primary cells and finite cultures ..85 Selecting a viral DNA delivery system ..86 Expression in mammalian cells ..86 vi | Cell culture basicsFor educational purposes for plasmid DNA transfection ..87 Vector considerations ..87 Quality of plasmid DNA ..87 Gene product and promoter.

9 88 Controls ..88 Optimization of plasmid DNA transfection ..89 Considerations for calcium phosphate coprecipitation ..89 Considerations for cationic lipid mediated delivery ..90 Considerations for electroporation ..93 Selection of stable transfectants ..94 Before starting ..94 Kill curve ..94 Selection workflow ..95 Selecting an RNAi strategy ..96siRNA vs. vector approaches ..96 Nonvector siRNA technologies ..97siRNA transfection ..99 Vector-mediated RNAi ..99 Guidelines for RNA transfection ..101 RNAi workflow ..101 Handling RNA ..102 Transfection efficiency ..102 Positive controls.

10 102 Negative controls ..103 Cotransfection ..103siRNA quality ..104siRNA quantity ..104 Volume of transfection reagent ..105 Cell density ..105 Exposure to transfection agent/siRNA complexes ..105 Presence of serum during transfection ..105 Tips for a successful siRNA experiment ..106 Optimization of siRNA transfection ..107 Factors affecting siRNA transfection efficiency ..107 Cell culture BASICS | viiFor educational purposes ..108 Troubleshooting ..108 Cell culture and transfection products ..109 Cell lines ..109 Media for mammalian cell culture ..110 Media for insect cell culture.