Growing and Studying Cells in Culture Flashcards
(25 cards)
How can you obtain a heterogeneous population of cells from tissue?
treat tissue with EDTA and proteases to disrupt intercellular attachments and form a heterogeneous population of cells
What does EDTA do?
EDTA is a calcium chelator that facilitates the release of cells from tissues by disrupting CAMs
What do proteases do?
Hydrolyze proteins (cleave protein-protein interaction) to release cells from tissues
What are CAMs?
- cell adhesion molecules
- allow cells to adhere to surface and each other
- need calcium to function
- disrupted by EDTA
What is a primary cell culture?
- cells prepared directly from the cells of an organism
- usually display the properties of the original tissue
- have a finite number of doublings (50-100) before they die
What is a cell strain?
a lineage of cells from one initial primary culture
What is a “passage of cells”?
subculture or splitting of confluent cells into a new culture once the culture has reached confluency
When have cells reached confluency?
- when they grow to cover the entire surface of the culture vessel
- are in contact with one another
What is contact inhibition?
- when cells stop growing when they are surrounded by other cells
- does not apply to cancer cells
What is a cell line?
- immortal cells that grow to higher densities
- derived from a tumor or have underdone spontaneous genetic change (oncogenic transformation)
What is a disadvantage of a cell line?
cells may not accurately represent the properties of the original cells due to the mutation
How are embryonic stem cells grown?
- Cleavage-stage embryo is developed by in-vitro fertilization
- Embryo develops into cultured blastocyst
- Inner cell mass removed from blastocyst (blastocyst now destroyed)
- Inner cell mass is placed in a petri dish of fibroblast feeder cells or cytokines
- Re-plate the dissociated cells in new feeder cells
- ES cell cultures are established
What does flow cytometry do?
- takes advantage of cell surface antigens to separate different cell types
- ex: T cells and red fluorescence and green fluorescence antibodies
- uses a fluorescence activated cell sorter (FACS)
How does a FACS work?
- scattered light detector detects cells based on size and granularity
- fluorescent light detector identifies fluorescently labeled cells
- fluorescent cells receive a negative charge
- drops with no charge are discarded
How should epithelial cells be grown to mimic how they are structured in tissues?
in a 2D culture with an apical medium and a basal medium
How are hybridomas produced?
- Inject mouse with antigen X
- Mouse spleen cells made antibody to antigen X
- Mouse spleen cells are fused with mouse myeloma cells
- Cells are transferred to a selective medium where unfused cells die and fused cells grow
- Culture single cells in separate wells
- Now have a permanent source for the monoclonal antibody from the hybridoma cells
What selective medium is used when producing hybridomas?
HAT (Hypoxanthine Aminopterin Thymidine)
provides aminopterin to block the De Novo pathway of DNA synthesis
What is the De Novo pathway of DNA synthesis?
Sugars + Amino Acids -> Nucleotides -> DNA
blocked by Aminopterin
What is the Salvage Pathway of DNA synthesis?
Nucleosides-> Nucleotides + DNA
need Tk and HGPRT
Why do unfused myeloma cells die on the HAT medium?
- they are unable to synthesize DNA
- need to go through De Novo pathway because they lack Tk and HGPRT
- De Novo pathway is blocked by Aminopterin
Why do hybridoma cells survive on the HAT medium?
- are immortal
- are able to synthesize DNA via the Salvage Pathway
Why do unfused B cells die on the HAT medium?
- they are mortal
- they are still able to synthesize DNA via the Salvage Pathway because they have Tk and HGPRT
What does PMSF do?
- phenylmethyl sulfonyl fluoride
- serine protease inhibitor
What does monastrol do?
- inhibits kinesin 5 (motor protein) that is necessary to separate the poles of the mitotic spindle
- tested as an anti-tumor drug in certain brain cancers
