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Cell Viability and Cytotoxicity

identifying live, dead, and injured cells
by

Leah Markham

on 22 April 2013

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Transcript of Cell Viability and Cytotoxicity

PC12 Cells, ECM and
Cell Viability & Cytotoxicity Cell line developed by Lloyd A. Green and Arthur S. Tischler Origin of PC12 Cells “PC” in PC12 comes from pheochromocytoma Origin of PC12 Cells Suspension vs. Adherent Have the ability to continually grow and divide Why do We Use PC12 Cells? Medulla Cortex PC 12 cells come from here Published results in April 1976 Outlined how cell line was created Outlined contents of media used 85% RPMI-1640, 10% HS, 5% FBS This is the same media we use Type of tumor Found in adrenal glands PC12 line comes from a rat adrenal medulla tumor Unlike primary neurons When exposed to NGF, they extend neuronal-like processes Can be made to produce neurotransmitters like norepinephrine and dopamine Extracellular Matrix -Complex network of polysaccharides and proteins secreted by cells; serves as a structural element in tissues and also influences their development and physiology Proteoglycans Collagens Major Components Definition: Family of fibrous proteins Long, stiff, triple-stranded helical structure 25 distinct types have been identified Each coded for by a different gene Enable ECM to resist stretching Major Components Made up of GAG chains covalently linked to a core protein GAGs enable the ECM to resist compression Regulate activities of secreted proteins Cell Viability and
Cytotoxicity Two main options: How to Test Viability/Cytotoxicity We need to evaluate whether the cells we culture are healthy Why we Test Viability/Cytotoxicity Replace culture media with PBS Live/Dead Viability Assay MTT is added to cell suspension and incubated for 2-4 hours MTT Assay Add stain to cell suspension Trypan Blue Exclusion Using two different stains, can evaluate both living and dead cells simultaneously Live/Dead Mammalian Viability Assay Indicates live, healthy cells MTT Assay viability cytotoxicity Definitions Stain penetrate membrane of dead cells Trypan Blue Exclusion 1. the degree to which an agent has specific destructive action on certain cells. 2. the possession of such destructive action, particularly in reference to lysis of cells by immune phenomena and to antineoplastic agents that selectively kill dividing cells. 1. capable of living, of normal growth and development Are cells more or less healthy on PLL/PDL than NM? We need a way to quantify this data Use a stain that will permeate membrane of dead cells, but not living cells Use a stain that healthy cells break down into a different compound
indicates intracellular esterase activity as well as membrane's ability to retain esterase products Using microscopy, plate readers, and/or flow cytometers can determine cell viability/cytoxicity Dead cells are stain blue Healthy cells remain unstained Can be evaluated using phase contrast microscopy Active cells metabolize MTT compound, producing a purple color Amount of color can be measured using a plate reader Evaluated using fluorescence microscopy Dead cell nuclei stained with Ethidium homodimer-1 (red) Living cell membranes stained with Calcein AM (green) Using phase contrast microscope, count total number of stained and unstained cells (# unstained cells)/(total # cells) = % viability Cell color evaluated using plate reader at 570nm Amount of color produced is directly proportional to viability of cells Add both stains to cell suspension Incubate 15-30 minutes Analyze using florescence microscopy (# green stain cells)/(total # stained cells)= % viability Looking Forward 7. Repeat assay tests on primary neurons 6. Use a second assay to retest all materials 4. Test cytotoxicity on NM at 3, 5,7, and 9 DPD 3. Test cytotoxicity on PLL at 3, 5, 7, and 9 DPD 2. Test cytotoxicity on PDL at 3, 5, 7, and 9 DPD 1. Test differentiation of PC12 cells on PLL vs. nano-material Background
on PC12 Cells Removed tumor from rats
Minced and titrated tumor
Washed cells in PBS and centrifuged to remove debris (3 cycles)
Resuspended in growth medium
Passaged on collagen-coated dishes
Mechanically detached from dish to separate from other types of cells Original paper suggested they would be useful for studying the initiation and regulation of neurite outgrowth How did We Choose the Media? The media we use is the same media used to create the PC12 cell line
Green and Tischler tested other medias and determined RPMI-1640 worked best
We don't use antibiotics because they can lead to antibiotic resistant strains
This also forces us to develop good sterile technique Why is Extracellular Matrix Important? Overall, the purpose of this research is to determine whether MSNF provides a suitable ECM for neurons
The MSNF mimics naturally occuring ECM in that it provides a 3-D scaffolding, rather than the 2-D surface provided by PDL or PLL Major Components Glycosaminoglycans (GAGs) Highly negatively charged
Form hydrated gels The negative charge attracts cations, which cause lots of water to be sucked into the matrix, creating a swelling pressure and allowing the matrix to resist compression Although they make up only a small part of ECM by weight, they take up the most volume, filling most of the space Healthy cells break Calcein Am down into Calcein, which then fluoresces green
Ethidium homodimer-1 permeates membranes of dead cells and binds to nucleic acids and fluoresces red
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