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 0.2µm • 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. 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

7. Microscopy of Live cells

8. 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

9. Absorb light at one wavelength and emit light at a specific and longer wavelength

10. HYDRA EXPRESSING GFP Fluorescent protein in live cells

11. FIX EMBED SECTION STAIN

12. 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

13. RAT INTESTINAL CELL WALL – GLUT 2

14. CONFOCAL AND DECONVOLUTIONMICROSCOPY • This overcomes the limitations of Fluorescence microscopy • Blurrred images • Thick specimens

15. REMOVES OUT OF FOCUS IMAGES

16. EXAMPLE OF IMAGE RECONSTRUCTED AFTER DECONVOLUTION MICROSCOPY

17. ELECTRON MICROSCOPY

18. 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

19. GOLD PARTICLES COATED WITH PROTEIN A ARE USED TO DETECT ANTIBODY BOUND TO PROTEIN

20. TEM IMAGE

21. 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

22. SURFACE DETAILS BY METAL SHADOWING

24. SEM OF EPITHELIUM LINING THEINTESTINAL LUIMEN

25. PURIFICATION OF CELL ORGANELLES • CELL DISRUPTION • SEPARATION OF DIFFERENT ORGANELLES USING CENTRIFUGATION • PREPARATION OF PURIFIED ORGANELLES USING SPECIFIC ANTIBODIES

26. BREAKING OPEN PLASMA MEMBRANES IN CELLS • CELLS ARE SUSPENDED IN ISOTONIC SUCROSE • SONICATION • HOMOGENIZATION • CELLS IN HYPOTONIC SOLUTION – RUPTURE OF CELL MEMBRANES

27. SEPERATING ORGANELLES • DIFFERENTIAL CENTRIFUGATION • DENSITY GRADIENT CENTRIFUGATION

29. DENSITY GRADIENT CENTRIFUGATION

30. ANTIBODIES ARE USED TO MAKE HIGHLY PURIFIED ORGANELLES

32. CELL SORTER – FLOW CYTOMETRY

33. 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,

34. 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

35. PRIMARY CULTURES

36. STAGES IN CELL CULTURE

37. DIFFERNTIATION OF A CELL LINE – C2C12 IN CULTURE

38. HOMEWORK-1 • CHAPTER 9 • REVIEW CONCEPTS QUESTIONS -2,5,7,9 • ANALYZE THE DATA DUE NEXT WEEK IN THE WORKSHOPS