level: Cell Culture
Questions and Answers List
Flashcards on the case and lecture on Cell Culture
level questions: Cell Culture
| Question | Answer |
|---|---|
| what are the three main cell types that can be cultured in a lab and what are their differences? | 1. primary cells are directly isolated from human/animal tissue. they represent the tissue and organisms characteristics well, but they have limited proliferative capacity. 2. transformed cells are cells that have a fast growth rate and are stable in maintenance and for cloning. they are usually formed by genetic manipulation, which can lead to non-physiological phenotypes. 3. self-renewing cells that are able to differentiate into other cell types, such as pluripotent stem cells. |
| what is the difference between primary cells and cell lines? | primary cells are directly derived from the patient and have limited proliferation cycles. cell lines are formed when normal cells are cultured and allowed to divide and expand in number, which is usually due to genetic modifications. cell lines are often derived from a tumour of the cell type of interest or by transforming and immortalising a primary cell with an oncogene. contrary to primary cells, cell lines can be maintained indefinitely in culture and are able to generate large number of cells. |
| what are the pros and cons of neuronal cell lines | pros: can be maintained indefinitely, can generate large amount of cells, ability to manipulate gene expression and control cellular environment. cons: genetic alterations can accumulate, mature neurons are non-dividing and cannot be subcultured, cell line neurons do not represent the full phenotype of a neuron, there is loss of synaptic pathways and normal anatomic relations. |
| what are things to be considered when working with cell cultures in the lab? | - some cell lines are categorized in a high Hazard Group and are pathogenic. - lab workers should wear personal protective equipment (PPE). - surfaces that come in contact with the cell culture media should be cleaned with 70% ethanol. - an aseptic environment is created using a biosafety cabinet. |
| what are the steps of immunofluorescence? | 1. fixation to preserve morphology and cellular architecture. 2. permeabilization in case plasma membrane is not yet disrupted by fixation, to give antibodies access to intracellular epitopes. 3. embedding in paraffin to fix the tissue (not needed for cells). 4. deparaffination and rehydration. 5. antigen retrieval to restore epitope-antibody reactivity through; - protease induced epitope retrieval; enzymes cleave protein cross-links - heat-induced epitope retrieval; heat and pressure is used 6. blocking to prevent binding of antibodies to non-target epitopes. 7. primary or secondary immunofluorescence. 8. detection using fluorescent microscope. |
| what is the difference between primary and secondary immunofluorescence? | in primary fluorescence, one antibody coupled to a fluorophore that binds to the epitope is used, while in secondary fluorescence, a primary antibody is used to bind to the epitope and a secondary antibody coupled to a fluorophore is used to bind to the primary antibody. primary immunofluorescence is quicker, but secondary immunofluorescence is more widely used since it is more sensitive. |
| what is the difference between immunofluorescence and immunohistochemistry? | immunofluorescence uses a fluorophore to detect the epitope. this means that a confocal or other fluorescent microscope has to be used for analysis. the fluorescence emission can directly be measured. immunohistochemistry does not use a fluorophore, but instead enzymes to detect the epitope. this bond is visualised by the reaction of the enzyme with the epitope and the substrate, and the change in stain/colour that produces. this means that the sample can be analysed using a light microscope. this technique is semi-quantitative since the absolute presence of the epitope cannot be measured. |
| what is transfection? what are the two subtypes? and what are the three ways of transfection? | transfection is the process of introducing foreign nucleic acids into eukaryotic cells to genetically modify the cell. transfection can be stable, meaning that the foreign nucleic acids are integrated in the host's genome or remain present in the host cell as an extra-chromosomal element. this means that the transgene remains expressed as the cell replicates. transfection can also be transient, meaning that the foreign nucleic acids are present in the host cell as a plasmid or as oligonucleotides. after a couple of cell replications, the transgene will be lost. the three ways of transfection are viral transfection, physical transfection and chemical transfection. |
| what is the process of viral transfection (transduction)? | a viral vector, protected by an envelope, carries foreign nucleic acids into its capsid. the virus interacts with the host cell using surface proteins on its envelope. once inside the cell, the virus releases its nucleic acids. viruses that are often used for transduction are retroviruses (RNAs), adenoviruses (double-stranded DNA) and adeno-associated viruses (single-stranded DNA). retroviruses contain the enzyme integrase which allows for stable transfection. |
| what are the different ways of physical transfection? | creating holes in the cell membrane to allow for nucleic acid entry using: - electroporation: an electrical voltage is used to increase cell membrane permeability - sonoporation: microbubbles techniques - laser irritation-assisted transfection: a laser beam magnet-assisted transfection: a magnetic force is used to guide nucleic acids across the membrane gene microinjection: a needle is used to puncture the cell membrane and inject the nucleic acids into the nucleus. |
| what is the main principle of chemical transfection? and which molecules are used? | the two main principles of chemical transfection are - that certain positively charged molecules surround the negatively charged nucleic acids, thereby shield it and allow for entry into the cell. molecules that can do this are calcium phosphate, dendrimers, cationic polymers, nanoparticles. - that positively charged lipids surround the negatively charged nucleic acids, enabling merging with the cell membrane and thereby inserting the nucleic acids into the cell. |
| how can efficiency of transfection be assessed? | - fluorescence microscopy with fluorescently tagged molecules - real time PCR to directly measure nucleic acid expression level - plasmid reporting system - flow cytometry to quantify numbers of fluorescently labelled transfected cells - immunostaining |
| what are the disadvantages of the different physical transfection techniques? | the use of high voltage in electroporation can lead to necrosis, apoptosis and permanent cell damage. both sonoporation and laser irradiation transfection carry the risk of damaging the cell membrane and leading to irreversible cell death. compared to these techniques, magnetofection is less destructive to the cell, however, this technique is less efficient. gene microinjection can prevent cell damage very precisely, but this requires trained personnel or robotic systems. |
| what are applications for bacterial and human/animal cell culture? | bacterial cell culture: diagnosis of infections, genetic manipulation, food and beverage production, detecting food contaminants, development of vaccines and therapeutics, epidemiological studies. human/animal cell culture: gene therapy, model systems, toxicity testing, cancer research, drug screening, vaccine production, genetic engineering. |
| how can plasmids be inserted into bacteria? | 1. competent cell preparation: pick a cell colony, culture it at 37ᵒC, prepare the cells at 4ᵒC, freeze cells at -70ᵒC. 2. transformation, e.g. through chemical or physical transformation. 3. recovery in an antibiotic-free medium to allow for expression of antibiotic resistance genes. 4. cell plating on agar plate to determine transformation efficiency. |
| what is the difference between (bacterial) culture on agar plates or in liquid suspension? | a suspension is used when you want to upscale your culture. an agar plate is used to; - select individual colonies from a mixed culture - to count the colony forming units within a sample - to transport strains from one lab to another |
| what is the transformation efficiency? and what is the transfection efficiency? | transformation efficiency: the total number of colonies divided by the amount of DNA that was used to transform the cell. transfection efficiency: the number of cells that express the antigen/protein of interest divided by the total number of cells. |
| what is the goal of transformation in bacteria compared to transfection humans? | the goal of transformation in bacteria is to produce multiple copies of a recombinant DNA molecule. the goal of transfection in humans is to introduce nucleic acids into eukaryotic cells. |
| what are the pros and cons of physical transfection? | pros: simple to perform, good for difficult to transfect cell types, reproducible results, no vector required, less dependent on cell type, rapid transfection of large number of cells. cons: requires special instruments, parameters must be carefully optimized, potential for high toxicity as well as cell damage and mortality. |
| what are the pros and cons of viral transfection (transduction)? | pros: high efficiency, works well in difficult cells, can be used for the generation of stable or transient cell lines. cons: immunogenicity and cytotoxicity, technically challenging and laborious, high costs, variations in infectivity, low packaging capacity. |