Cellular Biology

1. Cellular Biology School of Life Sciences Shaanxi Normal University 1

2. CHAPTER 2 THE TECHNOLOGIES USED IN CELLULAR BIOLOGY

3. Cell culture • cultured cells can be obtained in large quantity; • most cultures contain only a single type of cell; • many different cellular activities, including endocytosis, cell motility, cell division, membrane trafficking, and macromolecular synthesis, can be studied in cell culture; • cells can differentiate in culture; • cultured cells respond to treatment with drugs, hormones, growth factors, and other active substances.

4. Primary culture: The cells are obtained directly from the organism. Usually, primary culture can be continued with limited passage number (about 50 times). Secondary (Passaged) culture: Cell line culture. Tumor cells can be cultured for long time with almost unlimited passage times. Mass culture: Culture for tissue mass. It is not usually used in lab. Cloning culture: Cell culture to colonize cell. If you want to develop some special cell line, you have to culture cell clonally. Culture on special basement or prop stand: Cells will grow up on some special basement or matrix. Cell line: Cells are developed from one cell with some specific characters.

5. Basic Steps for Cell Culture Construction of primary cell culture or cell line Culture with medium and other help reagents Observation and property checking Change medium Experimental treatments and results examination Cell passage and cell line stock Data analysis

6. What you have to know for cell culture: • CO2 • Temperature • pH and ion concentration • No any contamination • Start a culture from a stock • Passage and cell line stock Dish 6-wells plate

7. Microscopes and Images Optical microscope Optical inverted microscope Fluorescence microscope Electron microscope Laser confocal scanning microscope Polarized light microscope Phase contrast microscope Cell image station

8. Simple upright light microscope

9. Optical microscopes

10. Fluorescence microscope

11. Laser confocal scanning microscope

12. An photo taken by LCSM

13. Mechanism about phase contrast microscope

14. Human blood cells viewed by bright-field and phase-contrast light microscopy. Arrow indicates a white blood cell. Formyl-met-leu-phe causes the white blood cell to spread out and become very thin. It becomes almost invisible by bright-field microscopy but can still be detected by phase-contrast microscopy

15. A photo taken by phase contrast microscope

16. Optical inverted microscope

17. Transmission Electron Microscope

18. A tissue section machine for transmission electron microscope

19. A photo about endoplasmic reticulum taken by transmission electron microscope

20. Scanning Electron Microscope

21. Human blood cells photo taken by scanning electron microscope

22. A freeze-etching photo about a cell

23. A microscope operation system

24. Digital image microscope system

25. Image station

26. Powerful microscopes: • Laser confocal scanning microscope (LCSM) • Catches a very thin focal plane within a thick specimen • Image at three different planes within the specimen without any damage to the specimen • Do not have to section tissue to get specimen slides for this microscope • Scanning tunneling microscope (STM) • Most powerful microscope in the world so far at atom grade • Stereoscopic surface image based on atomic grade • Specimen must be of electric conductivity • No any damage to the molecule’s function • Atomic force microscope (AFM) • Powerful also at atom grade • The specimen does not have to be of electric conductivity

27. Histochemistry, immunochemistry, display and other technologies • Isolation of the cell apparatus, biological macromolecules and complexes: Differential centrifugation The cell components will be divided following their different sedimentation coefficient, “S” Centrifuge methods to isolate cell components Density gradient centrifugation If the both methods are used together, you will obtain the better isolation result than using one method alone.

28. Differential centrifugation Using different RPM you will spin down the particles with different sedimentation coefficient

29. Density gradient centrifugation (A) Sedimenting with same speed; (B) Sedimenting with same density

30. 2. Display and stain the nucleic acids, protein, enzyme, sugar, and fat/lipid: Nucleic acids: Feulgen reaction (Schiff reaction). Sugar: PAS reaction. Fatty/Lipid: Sudan III or Sudan Black reaction. Protein: Millon reaction, Ninhydrin staining. Enzyme: Use ubstrate reaction or the methods same to stain protein as above. 3. Locate and quantitate the antigen in cells: Usually, we use HRP (Horseradish peroxidase), AP (Alkaline phosphatase), biotin or inflorescence to label the Ab against the specific Ab against the Ag you want to check or display. We call the labeled Ab as second Ab and the Ag binding Ab as first Ab.

31. The complex of Ag plus Ab The labeled Ab against the first Ab (Second Ab) The substrate for enzyme or biotin The cell, bacterial, virus, tissue or others with the Ag you want to check Enzyme, biotin, isotope or others The color, bright or radio ray particles that are visible by your eyes or machine The antigen you want to check or display from any bio-organisms, component, or other objects. The Ab against the Ag specificly (First Ab) Immunoenzyme methods to detect specific Ag or Ab ELISA, ELISPOT and others

32. Specific Enzymatic Detection of Membrane-Bound Antigens • Unoccupied sites on the membrane • Primary antibody to a specific antigen is incubated with the membrane • A blotting-grade antibody-enzyme conjugate is added to bind to the primary antibody • Color development reagent is added to the blot; the HRP or AP enzyme catalyzes the conversion of the substrate (S) to a colored precipitate (P) at the site of the antigen-antibody complex

33. Western blotting Western blotting is the most important and popularly used detection method for cell biology, molecular biology and molecular immunology to check gene expression, antigen, components of antigen, and antibody. The good western blotting performance skills are the basic requirements to every body who is working in a life science research laboratory. It is not so easy to get good data (images).

34. Basic steps for Western Blotting – Cathode – Cathode +Anode + Anode Develop each band of each lane to be visible with a serial steps as a image result Protein migration direction Gel NC membrane

35. Example results of Western Blotting

36. A model picture of the transfer electrophoresis of western blotting

37. A component of the Criterion precast gel system, the Criterion blotter combines blotting efficiency and flexibility in a unit that is incredibly easy to use

38. The Mini Trans-Blot cell provides rapid, high-quality blotting of mini gels. A component of the Mini-PROTEAN 3 electrophoresis system, the Mini Trans-Blot cell accommodates two gel holder cassettes for electrophoretic transfer of both mini format gels run in the Mini-PROTEAN 3 cell.

39. The Trans-Blot Plus cell offers rapid and effective transfers over a large 26.5 x 28 cm blotting area. Designed for use with large format gels, such as those used with the PROTEAN Plus Dodeca cell, this versatile tank transfer system provides uniform transfers in as little as 15–30 minutes

40. Bio-Rad offers two types of blotting apparatus that complement our vertical cells

41. The Bio-Dot and Bio-Dot SF (slot format) microfiltration units provide a reproducible method for binding protein or nucleic acid in solution onto nitrocellulose or Zeta-Probe membrane. Many experimental protocols can be accommodated by using interchangeable templates to form the 96-well Bio-Dot apparatus or 48-well slot format Bio-Dot SF apparatus. Each is available as a complete, independent unit or as a modular template without the manifold base

42. Mini incubation trays allow safe, simple, and economical screening of antigens that have been blotted onto membranes. With these trays, the entire Immun-Blot assay screening process can be carried out in the tray, minimizing exposure to biohazardous materials. Because the trays are disposable, concerns associated with washing reusable trays are eliminated

43. Using the Mini-PROTEAN II multiscreen apparatus, you can quickly and efficiently screen up to 40 different antibodies or sera without having to cut the western blot into individual strips. This unit clamps the blot securely in place, creating 40 leakproof channels. Individual samples can be screened without cross-contamination. Additionally, procedures like screening monoclonal antibodies and monitoring antibody titers from multiple sources are simplified

44. The Model 785 vacuum blotter quickly and efficiently transfers DNA or RNA from an agarose gel onto a nylon membrane. Because it generally requires only 90 min for transfer, the nucleic acid samples can be separated on a gel and transferred to a membrane, and the hybridization reaction begun on the same day

45. Northern transfer using the Model 785 vacuum blotter. Samples of 1.0, 2.5, 5, 10, and 20 µg of total RNA from CHO HA-1 cells were separated on a glyoxyl gel and transferred onto a Zeta-Probe membrane using 3" Hg for 90 min. The blot was probed with a 32P-labeled hsp70 cDNA fragment* and exposed to X-ray film overnight.

46. ELISA ELISA is always used to detect antigen or antibody in research lab or clinic lab because it is very easy, specific, and sensitive.

47. Immunofluorescence Technology Immunofluorescence Technology was designed based on the fluorescence labeled antibody technology. Stain the specific antigen, then you can view the stained antigen in cells or tissue under a fluorescence microscopy or take digital images as your experimental data. 4. Locate or quantitate the specific nucleic acid sequence in cells or tissue: Hybridization in situ is used for this object. Label specific oligonucleotide (DNA probe or RNA probe) with biotin, fluorescence, or isotope, make the probe hybridized with specific target sequence in cells or tissue, then check the samples under a fluorescence microscopy or electric microscope. Biotin, Isotope, or fluorescence Oligonucleotide probe Target DNA/RNA sequence

48. Human chromosome telomeres were displayed by Hybridization in situ with fluorescence labeled probe

49. An photo taken by fluorescence microscope Nucleus Micro filament Micro tubes

50. 5. Analyze the synthesis of the macromolecules in cells with radio labeling technology 3H inserting method: 1. One of the 4 dNTPs is labeled. Develop your sample as DNA/RNA image data. 2. One of the 20 amino acids to be labeled. Develop your sample as protein image data. 6. Flow cytometry (FCM) 1. To quantitate some molecule in or on cells 2. To check the cell subsets, immunity status 3. To sort cells into tubes for the culturing Usually, the antibody against some special marker (first antibody) on the cell surface is labeled by fluorescence.