david weisblat 385 life sciences addition phone 642-8309 email: [email protected]
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David Weisblat 385 Life Sciences Addition Phone 642-8309 Email: [email protected]. Office hours by appointment. Cell culture = propagation of cells outside the organism. Advantages: 1) cellular environment can be manipulated 2) cell type well defined - PowerPoint PPT PresentationTRANSCRIPT
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David Weisblat385 Life Sciences AdditionPhone 642-8309Email: [email protected]
Office hours by appointment
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Advantages:1) cellular environment can be manipulated2) cell type well defined3) large quantities of cells can be obtained4) many cellular functions can be investigated
Three types of cultured cells:1) primary cultures2) cell strains3) cell lines
Cell culture = propagation of cells outside the organism
Caveat: How well does what we see in the dish correspond to what goes on in the animal?
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dish of cell colonies
100 mm
single cell- scanning EM
0.01 mm
colony of cells
1 mm
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Primary Cultures
• Cells derived directly from tissues
First developed in 1907:
spinal cord explant+
lymphatic fluid
1 day
axons growin culture!
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cultured neuronextending processes
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Cell Strains
• Derived from primary cultures
tissue explant
dissociate cells
cells
plate cellsmedia
dish
• Grow and divide while adhering to plastic dishes
• Require media for growth
• Cells die after 50-100 divisions
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Cell LinesCan be derived from several different sources:
1) from cell strains (“normal cells”)• rare genetic changes generate variant cells thatcan grow indefinitely (immortal)
• example: BSC-1 cell line (derived from African Green Monkey kidney)
• grow until contacting neighboring cells, then exitcell cycle (experience contact inhibition)• will not form tumors when injected into mice
• cell strains• transformed cells• tumor cells
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Mouse Cell Line Derived from Cell Strain
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Some Cell Lines:
Retain properties of precursor cells:e.g. liver cell linesT-cell lines
Can differentiate in culture:e.g. epithelial cell linesmuscle cell lines
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differentiatingmusclecell line =C2C12
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Cell lines
2) from “transformed” cells• further genetic changes: can be caused by radiation, chemical carcinogens, tumor virusessee changed morphology, loss of growth control, loss of
contact inhibition• example: 3T3-21F• will form tumors in mice
3) from tumor cells• similar to transformed cells
• example: HeLA - from cervical carcinoma(Henrietta Lacks, 1951)
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normal transformed by virus
contact-inhibited pile up, rounded
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normal cells
transformed cells
scanning EM
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Properties of Cancer Cells
1. Lack normal growth controlsa) self-sufficiency in growth signalsb) insensitivity to anti-growth signalsc) evade programmed cell death (apoptosis)d) unlimited replicative potential
2. Able to invade tissues and metastasizea) loss of dependency on anchorage for growthb) loss of contact inhibition
3. Able to develop vasculature - blood supply“angiogenesis”
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Tumor Promoters
• Enhance tumor formation when combined with carcinogens, but are not themselves carcinogenic
carcinogentumor formation
+
tumor promoter -
carcinogen + tumorpromoter +++
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TPA = phorbal myristate acetate = PMA
• mimics 1, 2 diacylglycerol (DAG)
• DAG + Ca++ activates protein kinase C (PKC)
• causes phosphorylation of PKC substrates
• changes in cell growth, cell shape and the cytoskeleton
Phorbol ester
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Two cytoskeletal elements examined:
Examining cell architecture using fluorescence microscopy ?
• can visualize and localize individual proteins within a cell.
Test the effects of different drugs on the cytoskeletonand cell shape
TPA/PMALatrunculin
TaxolNocodazole
Actin Microtubules
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Actin structures in a fibroblast cell
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Microtubules = greenDNA= blue
interphase
mitosis
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Visualizing the cytoskeleton using fluorescence microscopy
1) Prepare samples: Fixation - kills and immobilizes cells
A. aldehydes - cross-link amino groups in proteins(formaldehyde, glutaraldehyde)
B. alcohols - denature proteins, precipitate in place(methanol)
Permeabilization - detergents make proteins accessible to staining reagents (Triton X100)
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2) Staining
Actin - phalloidin covalently linked to rhodamine(red) - binds to filamentous actin only
Microtubules - immunofluorescence1o ab: rabbit anti-tubulin; 2o ab: fluorescein anti-rabbit
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3) Fluorescence microscopy
excitation emission
fluorescent molecule
Fluorochrome Excitation wavelength Emission wavelengthFluoroscein 490 - blue 520 - greenRhodamine 550 – green 580 - redHoechst (stains DNA) 345 - UV 455 - blue
wavelength
ex em
inte
nsity
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Fluorescence microscope