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Lecture 3

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University of Toronto Scarborough
Biological Sciences
Rene Harrison

092711 Lecture 3Studying Cells ER GolgiCell CultureCell Culture Studying cells in controlled in vitro system Grown in plastic flasks in mediaWhy use cultured cells in research Cultured cells can be obtained in large quantity most cultures contain only a single type of cell wide variety of different cells can be grown in cultureHELA CELLS Why use cultured cells in research Many different cellular activities can be studied chance to study cell differentiation cultured cells respond to treatment w drugs hormones growth factors and other active substances y Stem CellPluripotent y To treat Juvenile diabetes Alzheimers Parkinsons spinal cord injuries etc BrightField Light Microscope Visualizing cells fix cells stain cells mount to slides tissue embed and section firstKNOCK OUT MOUSEOther Light Microscopes y Light Microscopes Useful in examination of intracellular components in cells fixed or living at relatively high resolution y ResolutionThe extent to which fine detail in specimen can be discriminated or resolveddepends on wavelength of light y ie dynamic motility of mitochondria mitotic xsomes 1 Phase Contrast and Differential Interference Contrast DIC For small unstainedspecimens like living cells want to increase contrast wo staining use specialized optics to increase contrastMicroscopyFluorescent Microscopy 2 applications immunofluorescence GFP technology 2 types of microscopes fluorescent microscope lamp light source confocal microscope laser light source y Fluorescent Microscopes Cell is stainedtransfected w fluorochromes Cell is illuminated w UV light fluorochromes absorb UV light and electrons release some energy as longer visible light wavelengthsAntibodies IgG Proteins made by B cells binds specifically to a protein antigen Antibody gene undergoes nucleotide rearrangements in B cells When IgG on B cell binds to specific antigen B cell proliferatesmakes huge among of same antibodymuch of which is secrete into bloodstream y Therefore for every B cell has a unique gene therefore makes unique IgG therefore will recognize unique foreign antigen y Making Antibodies to your Protein Often covalently like fluorochrome fluorescein or rhodamine to an antibodybinds to appropriate protein in the cellimmunofluorescence y ie human protein ie actinFluorescent Imaging Proteins
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