cell culture facilities and equipment
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Cell Culture Facilities and Equipment. Presenter: Dr. R. Rahbarghazi. Small Tissue Culture Laboratory suggested for use by two or three persons. Medium-Sized Tissue Culture Laboratory suitable for five or six persons. - PowerPoint PPT PresentationTRANSCRIPT
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Cell Culture Facilities and Equipment
Presenter: Dr. R. Rahbarghazi
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Small Tissue Culture Laboratory suggested for use by two or three persons
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Medium-Sized Tissue Culture Laboratory suitable for five or six persons
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Tissue Culture Lab with Adjacent prep room with medium-sized tissue culture lab
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Large Tissue Culture Laboratory; suitable for 20 to 30 persons
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High-Efficiency Particulate Air (HEPA)
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1. Laminar flow hood (Biological safety cabinet)2. CO2 incubator (for most cells)3. Inverted Microscope4. Pipette aid5. Aspiration pump6. Centrifuge7. Water bath8. Cold storage9. Cryopreservation
Equipment
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1. Sterilization equipment2. Balances3. Vortex4. Water purification5. pH meter6. Magnetic stirrer7. Micro pippettor8. Cell counter9. Video camera or CCD camera
Additional or Optimal Equipment
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Laminar flow hood (Biological safety cabinet)
1. Vertical mode
2. Horizontal mode
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1. Class I biosafety cabinet2. Class II biosafety cabinet3. Class III biosafety cabinet
Vertical laminar flow hoods
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1. Air is circulated away from operator2. 100% of air is exhausted3. Is optimal for radionuclides and volatile (toxic)
materials4. Dirty room air contaminates materials
Class I Biosafety Cabinet
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Type II-A1. A front access opening with a carefully maintained inward
flow 2. HEPA-filtered unidirectional airflow3. HEPA-filtered exhaust air to the lab (30%)4. 70% of the air re-circulated back into the laminar flow
hood5. Are not suitable for wok with radionuclides or volatile
materials
Class II Biosafety Cabinet
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Type II B1. Are conducted to exterior of building2. Air are not re-circulated within the
cabinets3. Suitable for work with radionuclides
and volatile materials
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1. Providing highest level of protection for both material and operator
2. Is totally enclosed3. Is under negative pressure4. 100% of air is exhausted
Class III Biosafety Cabinets
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CO2 and N2 incubators
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Inverted Microscope
Fluorescent inverted microscope
Inverted microscope
Stereo or dissecting microscope
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Confocal microscopy
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Pipette aid
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Aspiration (vacuum) pump
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Stirrer Flask
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Bioreactors
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Centrifuge
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Cold storage
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Cryopreservation
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Sterilization equipment
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Micro-filters
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Glass vacuum filtration
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Balances Vortex
Water bath
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Water Purification
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Flow cytometry integrates electronics, fluidics, computer, optics, software, and laser technologies in a single platform
Flow cytometry
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Advantages: 1. Provides individual measurements of
cell fluorescence and light scattering2. Enables us to individually sort or
separate subpopulations of cells
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Fluorescence Activation Process (or Immunofluorescence)
FITC FITC
FITC
FITCFIT
C
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
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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. StructuralDNA contentDNA base ratiosRNA content
2. FunctionalSurface and intracellular receptors.DNA synthesisDNA degradation (apoptosis)Cytoplasmic Ca++Gene expression
Intrinsic Extrinsic
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Flow Cytometry Applications
ImmunofluorescenceCell Cycle KineticsCell KineticsGeneticsMolecular BiologyAnimal Husbandry (and Human as well)MicrobiologyBiological OceanographyParasitologyBioterrorism
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FSC Correlates With Cell SizeSSC Correlates With Internal Complexity
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• 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)
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Magnetic-Activated Cell Sorting (MACS)
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flow.mp4