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CELL BIOLOGY TECHNIQUES Visualize cells - Microscopy Organelles – Fractionation of subcellular components Culturing cells Light Microscopy Light Microscopy • Resolution of 0.2µm • Magnification – objective and projection lens • Resolution – D = 0.61λ/N sin α Resolution is improved by using shorter wavelengths or increasing either N or α. BRIGHT FIELD PATH MICROSCOPY Visualize unstained living cells • Phase Contrast microscopy – Thin layers of cells but not thick tissues • Differential Interference contrast – Suited for extremely small details and thick objects – Thin optical section through the object Microscopy of Live cells Fluorescence Microscopy • Major Function: Localization of specific cellular molecules – example proteins • Major Advantages: – Sensitivity:“glow” against dark background – Specificity: immunofluorescence – Cells may be fixed or living • Fluorescent dyes or proteins (Flurochromes) – flurochromes may be indirectly or directly associated with the cellular molecule – Multiple flurochromes may be used simultaneously Absorb light at one wavelength and emit light at a specific and longer wavelength HYDRA EXPRESSING GFP Fluorescent protein in live cells FIX EMBED SECTION STAIN Immunofluorescence Microscopy and Specific Proteins • Fluorescently tagged primary anti body • Fluorescently tagged secondary antibody • Fluorescently labelled antibody to tagged proteins such as myc or FLAG RAT INTESTINAL CELL WALL – GLUT 2 CONFOCAL AND DECONVOLUTION MICROSCOPY • This overcomes the limitations of Fluorescence microscopy – Blurrred images – Thick specimens REMOVES OUT OF FOCUS IMAGES EXAMPLE OF IMAGE RECONSTRUCTED AFTER DECONVOLUTION MICROSCOPY ELECTRON MICROSCOPY • Transmission EM – theoretically 0.005 nm; practically 0.1 nm –1 nm (2000x better than LM) – High – velocity electron beam passes through the sample – 50-100 nm thick sections – 2-D sectional image – surface details are revelaed – Subcellular organelles • Scanning EM – Resolution about 10 nm – Secondary electrons released from the metal coated unsectioned specimen – 3-D surface image GOLD PARTICLES COATED WITH PROTEIN A ARE USED TO DETECT ANTIBODY BOUND TO PROTEIN TEM IMAGE CRYOELECTRON MICROSCOPY • HYDRATED, UNFIXED AND UNSTAINED SAMPLES • SAMPLES ARE OBSERVED IN ITS NATIVE HYDRATED STATE • METHOD - AN AQUEOUS SUSPENSION OF SAMPLE IS APLLIED ON A GRID AND HELP B Y A SPECIAL MOUNT • 5 nm RESOLUTION SURFACE DETAILS BY METAL SHADOWING SEM OF EPITHELIUM LINING THE INTESTINAL LUIMEN PURIFICATION OF CELL ORGANELLES • CELL DISRUPTION • SEPARATION OF DIFFERENT ORGANELLES USING CENTRIFUGATION • PREPARATION OF PURIFIED ORGANELLES USING SPECIFIC ANTIBODIES BREAKING OPEN PLASMA MEMBRANES IN CELLS • • • • CELLS ARE SUSPENDED IN ISOTONIC SUCROSE SONICATION HOMOGENIZATION CELLS IN HYPOTONIC SOLUTION – RUPTURE OF CELL MEMBRANES SEPERATING ORGANELLES • DIFFERENTIAL CENTRIFUGATION • DENSITY GRADIENT CENTRIFUGATION DENSITY GRADIENT CENTRIFUGATION ANTIBODIES ARE USED TO MAKE HIGHLY PURIFIED ORGANELLES CELL SORTER – FLOW CYTOMETRY CELL CULTURE REQUIREMENTS • SOLID MEDIA – Specially coated plastic dishes or flasks (CAMs’) – Agar as the medium GROWTH MEDIA Rich in nutrients- amino acids, vitamins, salts fatty acids, glucose, serum provides the different growth factors, TYPES OF CULTURED CELLS • PRIMARY CELL CULTURES – DIFFERENTIATE IN CELL CULTURE • CELL STRAIN – ALSO HAVE A FINITE LIFE SPAN (FROM A PRIMARY CULTURE) • CELL LINE - INDEFINITE LIFE SPAN PRIMARY CULTURES STAGES IN CELL CULTURE DIFFERNTIATION OF A CELL LINE – C2C12 IN CULTURE HOMEWORK-1 • CHAPTER 9 – REVIEW CONCEPTS QUESTIONS -2,5,7,9 – ANALYZE THE DATA DUE NEXT WEEK IN THE WORKSHOPS
