cell biology techniques
DESCRIPTION
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 = 0.61 λ /N sin α - PowerPoint PPT PresentationTRANSCRIPT
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CELL BIOLOGY TECHNIQUES
Visualize cells - MicroscopyOrganelles – Fractionation of
subcellular componentsCulturing cells
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Light Microscopy
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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.
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BRIGHT FIELD PATH MICROSCOPY
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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
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Microscopy of Live cells
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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
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Absorb light at one wavelength and emit light at a specific and longer wavelength
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HYDRA EXPRESSING GFP
Fluorescent protein in live cells
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FIXEMBEDSECTIONSTAIN
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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
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RAT INTESTINAL CELL WALL – GLUT 2
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CONFOCAL AND DECONVOLUTIONMICROSCOPY
• This overcomes the limitations of Fluorescence microscopy– Blurrred images– Thick specimens
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REMOVES OUT OF FOCUS IMAGES
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EXAMPLE OF IMAGE RECONSTRUCTED AFTER DECONVOLUTION MICROSCOPY
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ELECTRON MICROSCOPY
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• 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
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GOLD PARTICLES COATED WITH PROTEIN A ARE USED TO DETECT ANTIBODY BOUND TO PROTEIN
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TEM IMAGE
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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
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SURFACE DETAILS BY METAL SHADOWING
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SEM OF EPITHELIUM LINING THEINTESTINAL LUIMEN
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PURIFICATION OF CELL ORGANELLES
• CELL DISRUPTION• SEPARATION OF DIFFERENT ORGANELLES
USING CENTRIFUGATION• PREPARATION OF PURIFIED ORGANELLES
USING SPECIFIC ANTIBODIES
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BREAKING OPEN PLASMA MEMBRANES IN CELLS
• CELLS ARE SUSPENDED IN ISOTONIC SUCROSE• SONICATION• HOMOGENIZATION• CELLS IN HYPOTONIC SOLUTION – RUPTURE
OF CELL MEMBRANES
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SEPERATING ORGANELLES
• DIFFERENTIAL CENTRIFUGATION• DENSITY GRADIENT CENTRIFUGATION
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DENSITY GRADIENT CENTRIFUGATION
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ANTIBODIES ARE USED TO MAKE HIGHLY PURIFIED ORGANELLES
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CELL SORTER – FLOW CYTOMETRY
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CELL CULTURE REQUIREMENTS
• SOLID MEDIA– Specially coated plastic dishes or flasks (CAMs’)– Agar as the mediumGROWTH MEDIA
Rich in nutrients- amino acids, vitamins, salts fatty acids, glucose, serum provides the different growth factors,
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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
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PRIMARY CULTURES
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STAGES IN CELL CULTURE
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DIFFERNTIATION OF A CELL LINE – C2C12 IN CULTURE
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HOMEWORK-1
• CHAPTER 9– REVIEW CONCEPTS QUESTIONS -2,5,7,9– ANALYZE THE DATA
DUE NEXT WEEK IN THE WORKSHOPS