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

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

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

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

Large Tissue Culture Laboratory; suitable for 20 to 30 persons

High-Efficiency Particulate Air (HEPA)

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

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

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

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

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

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

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

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

CO 2 and N 2 incubators

Inverted Microscope Fluorescent inverted microscopeInverted microscope Stereo or dissecting microscope

Confocal microscopy

Pipette aid

Aspiration (vacuum) pump

Stirrer Flask

Bioreactors

Centrifuge

Cold storage

Cryopreservation

Sterilization equipment

Micro-filters

Glass vacuum filtration

Balances Vortex Water bath

Water Purification

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

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

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

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

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

FSC Correlates With Cell Size SSC Correlates With Internal Complexity

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)

Emission Spectra Excitation Spectra

Magnetic-Activated Cell Sorting (MACS)