chapter 1 Flashcards
List 4 reasons to quantitate cells.
1) Maintenance
2) Consistency
3) Reproducibility
4) Cell characterization
What are two methods to quantitate cells and explain the advantages and disadvantages of each.
1) Hemocytometer
Advantages: Easy, cheap
Disadvantages: Takes long, Error prone
2) Electronic Cell Counter
Advantages: Fast, no staining
Disadvantages: Expensive
What does Transfection mean?
Adding foreign DNA/RNA to eukaryotic cells
Explain the two types of transfection?
1) Transient: DNA/RNA not integrated into chromosome. Not hereditary, high levels but dont last long.
2) Stable: permanent, DNA integrated into chromosomes, rare efficiency
Name a BIOLOGICAL transfection method and explain the advantages & disadvantages.
Virus mediated.
Advantages: high efficiency, easy to achieve transfection, can be done in-vivo or in-vitro
Disadvantages: inflammatory reactions, mutations, expensive, low packaging capacity.
Explain the three PHYSICAL transfection methods and list the advantages & disadvantages
1) Direct Injection: Insert DNA into cell with a needle
Advantages:
Disadvantages: requires skill, labour intensive, can cause cell death
2)Electroporation: short electrical pulses to cell membrane creates small holes for nucleic acid to pass through.
Advantages: most widely used, fast and efficient
Disadvantages: Can cause cell death
3) Laser Irradiation: Laser makes temporary hold and nucleic acid enters due to osmosis
Advantages: pores can be made at specific locations, can be done on very small cells
Disadvantages: expensive
Explain the two CHEMICAL transfection methods and list the advantages & disadvantages
1) Calcium Phosphate: DNA is mixed with calcium chloride and phosphate buffer which forms a precipitate that is taken up by cells. Cheap, highly efficient, easy to use. Disadvantage: different cell types = different efficiency.
2) Cationic Lipids: +ve DNA lipids are attracted to -ve cell membrane. Cell is then taken in by endocytosis/phagocytosis
Advantages: easy, no size limit, high efficiency
Disadvantages: varied results between cells
Define centrifugation.
speeds up rate of sedimentation by spinning sample at high speeds. Allows for separation and isolation of cellular components.
What determines the rate of sedimentation?
RCF, media viscosity, particle size, density between particle and media.
What is differential centrifugation and what products will you have at the end?
A series of centrifuging, increasing the speed with each run. You will have a pellet and supernatant.
What is Density Gradient Centrifugation?
Separates sample based on rate of sedimentation. Forms different layers for different densities. Sucrose is usually used.
Name the two types of centrifuge rotors.
1) Swinging Bucket
Fixed Angle
Define Cytometry
The measurement of physical and chemical characteristics of cells
What is a Flow Cytometer and list the advantages compared to using a microscope smear.
Used to measure and analyze physical characteristics of a particle/cell through a beam on light.
faster, can count more cells, obtains more detailed information.
What can a Flow Cytometer measure?
cell size, volume, surface area, surface proteins, DNA, enzymes, proteins
What is the difference between Forward Scatter, Side Scatter and fluorescence?
Forward Scatter: Used to detect the size of the cell
Side Scatter: Used to determine cellular complexity
Fluorescence: used to determine physiological and chemical properties of a cell
How does a computer store flow cytometry data? Explain the two ways.
1) Histogram: only single parameter is plotted. Further along x = higher intensity
2) Dot Plot: shows 2 parameters plotted against each other. The further on x and higher on Y = greater intensity
List some of the advantages and disadvantages of Tissue Cultures.
Advantages: control of environment, reduces animal use, validation (known origin, purity etc)
Disadvantages: training needed, cost, contamination
List two ways to avoid Cross Contamination.
1) only use reputable cells from a cell bank
2) Only work with one cell line at a time
What is tissue engineering?
growing tissue for regenerative medicine applications
explain the process of tissue engineering.
1) design and construct a scaffold.
2) scaffolds are seeded with living cells that will grow over the scaffold.
3) Scaffold are then bathed in a nutrient rich media
4) When implanted into the body, cell layers build up over the scaffold.
Why are stem cells important?
stem cells can develop into a variety of different cells.
