1. Cell Culture Facilities and Equipment Presenter: Dr. R. Rahbarghazi

2. Small Tissue Culture Laboratory Small Tissue Culture Laboratory suggested for use by two or three persons

3. Medium-Sized Tissue Culture Laboratory suitable for five or six persons

4. Tissue Culture Lab with Adjacent prep room with medium-sized tissue culture lab

5. Large Tissue Culture Laboratory; suitable for 20 to 30 persons

6. High-Efficiency Particulate Air (HEPA)

7. 1. Laminar flow hood (Biological safety cabinet) 2. CO2 incubator (for most cells) 3. Inverted Microscope 4. Pipette aid 5. Aspiration pump 6. Centrifuge 7. Water bath 8. Cold storage 9. Cryopreservation Equipment

8. 1. Sterilization equipment 2. Balances 3. Vortex 4. Water purification 5. pH meter 6. Magnetic stirrer 7. Micro pippettor 8. Cell counter 9. Video camera or CCD camera Additional or Optimal Equipment

9. Laminar flow hood (Biological safety cabinet) 1. Vertical mode 2. Horizontal mode

10. 1. Class I biosafety cabinet 2. Class II biosafety cabinet 3. Class III biosafety cabinet Vertical laminar flow hoods

11. 1. Air is circulated away from operator 2. 100% of air is exhausted 3. Is optimal for radionuclides and volatile (toxic) materials 4. Dirty room air contaminates materials Class I Biosafety Cabinet

12. Type II-A 1. A front access opening with a carefully maintained inward flow 2. HEPA-filtered unidirectional airflow 3. HEPA-filtered exhaust air to the lab (30%) 4. 70% of the air re-circulated back into the laminar flow hood 5. Are not suitable for wok with radionuclides or volatile materials Class II Biosafety Cabinet

13. Type II B 1. Are conducted to exterior of building 2. Air are not re-circulated within the cabinets 3. Suitable for work with radionuclides and volatile materials

14. 1. Providing highest level of protection for both material and operator 2. Is totally enclosed 3. Is under negative pressure 4. 100% of air is exhausted Class III Biosafety Cabinets

16. CO 2 and N 2 incubators

17. Inverted Microscope Fluorescent inverted microscopeInverted microscope Stereo or dissecting microscope

19. Confocal microscopy

20. Pipette aid

23. Aspiration (vacuum) pump

26. Stirrer Flask

27. Bioreactors

28. Centrifuge

29. Cold storage

30. Cryopreservation

31. Sterilization equipment

32. Micro-filters

33. Glass vacuum filtration

34. Balances Vortex Water bath

35. Water Purification

38. Flow cytometry integrates electronics, fluidics, computer, optics, software, and laser technologies in a single platform Flow cytometry

39. Advantages: Advantages: 1. Provides individual measurements of cell fluorescence and light scattering 2. Enables us to individually sort or separate subpopulations of cells

40. Fluorescence Activation Process (or Immunofluorescence) FITC Antibodies recognize specific molecules in the surface of some cells, especially stem cells But not others When the cells are analyzed by flow cytometry the cells expressing the marker for which the antibody is specific will manifest fluorescence. Cells who lack the marker will not manifest fluorescence Antibodies are artificially conjugated to fluorochromes (PE, FITC, RH, Alexa flour, … Antibodies

41. Cellular Parameters Measured by Flow No reagents or probes required (Structural) Cell size (Forward Light Scatter) Cytoplasmic granularity (90 degree Light Scatter) Photsynthetic pigments Reagents are required 1. Structural 1. Structural DNA content DNA base ratios RNA content 2. Functional 2. Functional Surface and intracellular receptors. DNA synthesis DNA degradation (apoptosis) Cytoplasmic Ca++ Gene expression IntrinsicExtrinsic

42. Flow Cytometry Applications Immunofluorescence Cell Cycle Kinetics Cell Kinetics Genetics Molecular Biology Animal Husbandry (and Human as well) Microbiology Biological Oceanography ParasitologyBioterrorism

45. FSC Correlates With Cell Size SSC Correlates With Internal Complexity

47. Fluorochromes Are Molecules That Emit Fluorescence Upon Excitation With Light –FITC (Fluorescein Isothiocyanate) –PE (Phycoerythrin) –PerCP (Peridinin Chlorophyll Protein) –APC (Allophycocyanin) Some Fluorochromes Are Proteins, Some Are Small Organic Compounds –Ex. PE (Phycoerythrin)-Protein –Ex. FITC (Fluorescein Isothiocyanate)

48. Emission Spectra Excitation Spectra

57. Magnetic-Activated Cell Sorting (MACS)