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Cell biology techniques

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Learn about Cell biology techniques

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Cell biology techniques

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

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