1. Javad Jamshidi Fasa University of Medical Sciences, December 2015 Tools and Techniques for Studying Cells

2. Cell Culture Flow Cytometry Microscopy Centrifugation Gel Electrophoresis Blotting 2

3. In 1655, Robert Hooke used a primitive microscope and coined the term cells. As study of cells in intact animals and plants is difficult, molecular cell biologists often conduct experimental studies on cells isolated from an organism Culture of unicellular bacterial, fungal, or protist cells is easier than animal cells come from multicellular organisms 3

4. Closely as possible the conditions within an intact organism Temperature pH Ionic strength Access to essential nutrients The nine amino acids (phe, val, thr, trp, ile, met, leu, lys, his) Antibiotics Most animal cell types will grow only attached to a solid surface 4

5. Tissue fragments are treated with a combination of a protease (e.g., trypsin, collagenase, or both) and a divalent cation chelator The released cells are then placed in dishes in a nutrient-rich, serum- supplemented medium Fibroblasts usually divide more rapidly than other cells 5

6. 6

7. Cells derived from some tumors, cell line The Hela cell line, the first human cell line established, was originally obtained in 1952 from a malignant tumor (carcinoma) of the uterine cervix Cells in immortalized lines often have chromosomes with abnormal DNA sequences Growth of Cells in Two-Dimensional and Three- Dimensional Culture Mimics the In Vivo Environment 7

8. 8 Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream Measures cell properties and sorting them

9. 9

10. Light microscopy : uses visible light to observe specimens Bright-field microscopy Dark-field microscopy Phase-contrast microscopy Fluorescence microscopy Electron Microscopy: Uses electrons instead of light Transmission electron microscope (TEM) Scanning Electron Microscopy (SEM) 10

11. 11

12. 12 Perhaps the most versatile and powerful technique for localizing molecules within a cell by light microscopy Uses UV light and florescence dye

13. 13 Uses electrons instead of light The shorter wavelength of electrons gives greater resolution The limit of resolution is theoretically 0.005 nm The effective resolution in the study of biological systems is 0.1 nm, still about 2000 times better than with conventional high-resolution light microscopes Transmission Electron Microscope (TEM) Scanning Electron Microscopy (SEM)

14. 14

15. 15

16. 16

17. 17

18. 18

19. 19

20. 20 The principle behind centrifugation is that two particles in suspension with different densities will settle at different rates Centrifugation is used for two basic purposes: As a technique to separate one type of material from others As an analytical technique to measure physical properties (e.g., molecular weight, density, shape) of macromolecules. The sedimentation constant, s, is a measure of sedimentation rate.

21. 21

22. 22

23. 23

24. 24 An analytical method based on movement of charged particles because of an external electric field To separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments Velocity of a particle depends on the: size, shape and charge of the particle given applied voltage

25. 25 Agarose Are easily cast and handled Are typically run horizontally DNA fragments ranging from 50 base pair to several megabases Polyacrylamide Handling is more difficult Are run in a vertical configuration Higher resolving power for DNA 1bp

26. 26

27. 27 Horizontal Electrophoresis Vertical Electrophoresis

28. 28 Horizontal ElectrophoresisVertical Electrophoresis

29. 29 Is a method of transferring proteins, DNA or RNA, onto a carrier Southern blot for DNA Northern blot for RNA Western blot for Proteins

30. 30