Cell biology techniques

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    19-Jun-2015
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Transcript of Cell biology techniques

  • 1. CELL BIOLOGY TECHNIQUES Visualize cells - Microscopy Organelles Fractionation of subcellular components Culturing cells

2. Light Microscopy 3. Light Microscopy Resolution of 0.2m Magnification objective and projection lens Resolution D = 0.61/N sin Resolution is improved by using shorter wavelengths or increasing either N or . 4. BRIGHT FIELD PATH MICROSCOPY 5. 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 6. Microscopy of Live cells 7. 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 8. Absorb light at one wavelength and emit light at a specific and longer wavelength 9. HYDRA EXPRESSING GFP Fluorescent protein in live cells 10. FIX EMBED SECTION STAIN 11. 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 12. RAT INTESTINAL CELL WALL GLUT 2 13. CONFOCAL AND DECONVOLUTION MICROSCOPY This overcomes the limitations of Fluorescence microscopy Blurrred images Thick specimens 14. REMOVES OUT OF FOCUS IMAGES 15. EXAMPLE OF IMAGE RECONSTRUCTED AFTER DECONVOLUTION MICROSCOPY 16. ELECTRON MICROSCOPY 17. 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 18. GOLD PARTICLES COATED WITH PROTEIN A ARE USED TO DETECT ANTIBODY BOUND TO PROTEIN 19. TEM IMAGE 20. 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 21. SURFACE DETAILS BY METAL SHADOWING 22. SEM OF EPITHELIUM LINING THE INTESTINAL LUIMEN 23. PURIFICATION OF CELL ORGANELLES CELL DISRUPTION SEPARATION OF DIFFERENT ORGANELLES USING CENTRIFUGATION PREPARATION OF PURIFIED ORGANELLES USING SPECIFIC ANTIBODIES 24. BREAKING OPEN PLASMA MEMBRANES IN CELLS CELLS ARE SUSPENDED IN ISOTONIC SUCROSE SONICATION HOMOGENIZATION CELLS IN HYPOTONIC SOLUTION RUPTURE OF CELL MEMBRANES 25. SEPERATING ORGANELLES DIFFERENTIAL CENTRIFUGATION DENSITY GRADIENT CENTRIFUGATION 26. DENSITY GRADIENT CENTRIFUGATION 27. ANTIBODIES ARE USED TO MAKE HIGHLY PURIFIED ORGANELLES 28. CELL SORTER FLOW CYTOMETRY 29. 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, 30. 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 31. PRIMARY CULTURES 32. STAGES IN CELL CULTURE 33. DIFFERNTIATION OF A CELL LINE C2C12 IN CULTURE