BE115+-+Lecture03 - Lecture 3 Analysis of 2D Cell Culture...

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Lecture 3 Analysis of 2D Cell Culture “There is nothing like looking, if you want to find something. You certainly usually find something, if you look, but it is not always quite the something you were after.” - J.R.R. Tolkien 1
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Culture Conditions (experimental input) Purpose: to see how cells behave under a certain set of conditions (as discussed last week) Cell type Nutrients (high/low glucose, amino acids, etc.) Matrix (TC polystyrene, collagen, fibronectin, etc.) Additives (growth factors, hormones, antibiotics, poisons, etc.) Mechanical stresses Presence/absence of other cells 2
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Oski Challenge Because Oski loves science, he has decided to donated some of his muscle cells, lung cells, skin cells and nerve cells for basic research. Question: what cell behavior do we look for?
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Experimental outputs for cells Stasis, Division, and Apoptosis (death) Migration Attachment, Spreading, and Polarization Differentiation Intracellular structure Proteins Gene Expression Once we have the cell behaviors down, we can start designing devices and worrying about their properties 4
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Tissue Characterization ECM characterization Composition Histology SEM Birefringence FTIR Development Mechanical testing Medical imaging 5
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Where are we now? Week 2 - 2D and 3D Culture (input) Week 3 – 2D analysis Week 4 – 3D analysis Week 5 - Protein quantification (output/prep for output) Week 6 & 7 - SDS-PAGE & Western (output) 6
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Quiescence, Proliferation, or Death Are your cells growing, dying, or just hangin’? 7
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Growth Cell counting (by microscopy, Coulter counter, etc.) DNA synthesis or quantification (BrdU incorporation, fluorometric assays, etc.) Metabolic assays (MTT, XTT, etc.) Assays for cell cycle markers (cyclin dependent kinases, PCNA, etc.) 8
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Doubling time Can be quantified as a growth rate, but the math is tricky More often expressed as Doubling Time N(t) = N(t0)2(t-t0)/D N(t) = Number of cells at time t N(t0) = Number of cells at time t0 t = end of experiment t0 = beginning of experiment t-t0 = time the cells have had to grow D = Doubling time 9
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Passage Number vs. Doublings Passage number is just the number of times a culture has been passaged Passage 34 cells (sometimes written º34 or P34) have gone
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Spring '11 term at City College of San Francisco.

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BE115+-+Lecture03 - Lecture 3 Analysis of 2D Cell Culture...

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