MMSC451: Cell Culture Final

What is the purpose of a horizontal laminar flow hood?

protects cultures, not the researcher

What is the purpose of vertical laminar flow hood?

protects cultures and the research

What is a HEPA filter?

high efficiency particulate air filter

What is the purpose of incubators?

mimic the environment cells experience in vivo

What environmental factors are replicated in incubators for cell cultures?

temperature(basic insulation or water jacket), pH(sodium bicarbonate and CO2), and high humidity

What is the purpose of waterbaths?

warm media, media components, and to thaw cells

What is the definition of cell culture?

in vitro cultivation of organs, tissue, or cells outside of the natural environment and under controlled conditions

What are the basal media components used?

- balanced salt

- amino acid and vitamins: growth, function and division

-glucose: source of carbon

What is serum in cell culturing?

supplement that contains most growth factors, attachment and spreading factors, protease inhibitors, hormones, etc.

What is trypan blue?

an exclusion dye for assessing health/viability of cells. gets pumped out of living cells and stains dead cells

What is the equations used for cell counting?

(# of living cells/4) x dilution factor x 10^4

What is the Baculovirus?

- gene delivery system in which a virus is used to infect insect cells

- only infectious to insect invertebrates

- closed, circular, ds 134 kB genome

What does the baculovirus infect specifically?

lepidopteran(butterflies and moths)

What cell lines are used with Baculovirus?

- sf9 + sf21( pupal ovarian tissue of fall army worm)

- Hi5(ovarian cells of cabbage looper)

What is the process of EGFP baculovirus expression system?

-recombinant viral DNA + bacmid generation

- insect transfection

-virus amplification

- protein purification

What are the three methods to dislodge monolayers in adherent cell culture?

- sloughing

- trypsinization

- tapping flask

What confluency should cells be cryopreserved at?

90% viable and 80-90% confluency

What are the components of media for mammalian cell culture?

DMEM-FBS, FBS, P/S, glutamine

What is a primary culture?

derived from intact or dissociated tissues or organ fragments?

What are HEK293 cells derived from?

normal human embryonic kidney cells transformed via adenovirus

What is transfection?

process of introducing DNA into cells on purpose

What are the different methods of transfection?

- chemical

- lipid

- electroporation

- injection

Lipid Transfection

Positively charged lipids form liposome like structure with the negatively charged DNA. Liposome/DNA complexes fuse with the plasma membrane and enter the cell via endocytosis. Once in the cytoplasm DNA escapes the complex and enters the nucleus for expression(e.g. lipofectamine)

When is the best phase to use cultured cells?

mid-log phase with 97% viability

What is an explant?

living tissue transferred from plant to artificial medium for culture

What is a callus?

mass of irregular, undifferentiated, parenchymatous and totipotent cells

What is auxin?

plant hormone that promotes cell division, stem, and root growth

What is cytokinin?

plant hormone that promotes cell division or cytokinesis in plant roots and shoots

What are the optimal conditions for plant cultures?

- 16 hrs of light, 8 hrs in darkness

- temperature(15-30C)

- humidity

- pH of 5.5-5.8

What are VLPs?

Virus-like particles are multiprotein structures that mimic native viruses but lack the viral genome. They are non-infectious and non-replicating

How are VLPs formed?

budding of protein; structure consists of protein core, lipid bilayer, and membrane proteins