chapter 1

1) Maintenance 2) Consistency 3) Reproducibility 4) Cell characterization

1) Hemocytometer Advantages: Easy, cheap Disadvantages: Takes long, Error prone 2) Electronic Cell Counter Advantages: Fast, no staining Disadvantages: Expensive

Adding foreign DNA/RNA to eukaryotic cells

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

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.

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

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

speeds up rate of sedimentation by spinning sample at high speeds. Allows for separation and isolation of cellular components.

RCF, media viscosity, particle size, density between particle and media.

A series of centrifuging, increasing the speed with each run. You will have a pellet and supernatant.

Separates sample based on rate of sedimentation. Forms different layers for different densities. Sucrose is usually used.

1) Swinging Bucket Fixed Angle

The measurement of physical and chemical characteristics of cells

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.

cell size, volume, surface area, surface proteins, DNA, enzymes, proteins

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

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

Advantages: control of environment, reduces animal use, validation (known origin, purity etc) Disadvantages: training needed, cost, contamination

1) only use reputable cells from a cell bank 2) Only work with one cell line at a time

growing tissue for regenerative medicine applications

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.

stem cells can develop into a variety of different cells.