Flashcards in Biotech Final - Lab 7 Deck (21)
What are the two major advantages of using cell culture?
1) Ability to control the physiological environment of the cells (pH, temperature, oxygen, etc...
2) Ability to modulate the intra and extracellular physiological conditions of the cell
Mammalian cell culture techniques must be carried out using ________ techniques.
What are the limitations of cell culture?
1) More easily contaminated by microbes
2) only pg amount of proteins can be generated
3) Altered cellular properties over time due to passage
What is a passage?
When a cell culture becomes confluent, it is split by enzymatic digestion and an aliquot is placed into a new flask containing fresh media.
With each passage, what is altering the phenotypes of cells?
What are the different cell cultures that are available?
1) Organ culture
2) Primary explant culture
3) Mammalian cell culture
What is organ culture?
Derived from a specific organ in which the characteristics of the tissue in-vivo are retained
What is a primary explant culture?
A fragment of tissue is placed between a glass and liquid interface where it attaches.
What is mammalian cell culture?
An outgrowth of the primary explant culture and is dispersed into a cell suspension (either as a monolayer or free floating in the media suspension)
What cell line did we used?
How are microbes typically introduced?
By the researcher
What aseptic techniques must be carried out in cell culture?
1) Check the culture for contamination
2) The culture should contain antibiotic to remove any cryptic contamination
3) The reagents are sterile
4) The bottles used should not be used for the maintenance of other cell lines
5) Sterile techniques used at all times
What does a laminar flow hood consist of?
A front opening, sash, exhaust HEPA filter, rear plenum, supply HEPA filter, mechanical blower
What was the primary purpose of this lab?
To ectopically express PTEN in mammalian cells.
What is lipid-mediated transfection?
Polycationic lipids are used to create artifical membrane vesicles (liposomes) that have the ability to directly interact with the plasma membrane of a cell or, be taken up by non-receptor mediated endocytosis.
What is the cytotoxicity of lipid-mediate transfection?
Varies, but is generally low.
What cell types are competent to lipid-mediated transfection?
1) Adherent cells
2) Primary cell lines
3) Suspended cultures
Why are cationic lipids used?
Cationic lipids react spontaneously with DNA to form a unilamellar shell which can fuse to cell membranes.
Why are polycationic lipids used?
The majority of lipofection reagents consist of a mixture of what?
1) Synthetic cationic lipid
2) Fusogenic lipid