Transcript Cell Culture Principles and Techniques

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
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)
Equipment
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Laminar flow hood (Biological safety cabinet)
CO2 incubator (for most cells)
Inverted Microscope
Pipette aid
Aspiration pump
Centrifuge
Water bath
Cold storage
Cryopreservation
Additional or Optimal Equipment
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Sterilization equipment
Balances
Vortex
Water purification
pH meter
Magnetic stirrer
Micro pippettor
Cell counter
Video camera or CCD camera
Laminar flow hood (Biological safety cabinet)
1. Vertical mode
2. Horizontal mode
Vertical laminar flow hoods
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Class I biosafety cabinet
Class II biosafety cabinet
Class III biosafety cabinet
Class I Biosafety Cabinet
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Air is circulated away from operator
100% of air is exhausted
Is optimal for radionuclides and volatile (toxic)
materials
Dirty room air contaminates materials
Class II Biosafety Cabinet
Type II-A
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A front access opening with a carefully maintained inward flow
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HEPA-filtered unidirectional airflow
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HEPA-filtered exhaust air to the lab (30%)
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70% of the air re-circulated back into the laminar flow hood
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Are not suitable for wok with radionuclides or volatile materials
Type II B
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Are conducted to exterior of building
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Air are not re-circulated within the cabinets
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Suitable for work with radionuclides and
volatile materials
Class III Biosafety Cabinets
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Providing highest level of protection for both material and operator
Is totally enclosed
Is under negative pressure
100% of air is exhausted
CO2 and N2 incubators
Inverted Microscope
Inverted microscope
Stereo or dissecting
microscope
Fluorescent inverted 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
Flow cytometry integrates electronics, fluidics, computer, optics, software,
and laser technologies in a single platform
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Advantages:
Provides individual measurements of cell
fluorescence and light scattering
Enables us to individually sort or separate
subpopulations of cells
Fluorescence Activation Process
(or Immunofluorescence)
Antibodies recognize specific
molecules in the surface of some
cells, especially stem cells
FITC
Antibodies are artificially
conjugated to fluorochromes (PE,
FITC, RH, Alexa flour , …
FITC
Antibodies
FITC
FITC
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
Cellular Parameters Measured by Flow
Intrinsic
No reagents or probes required
(Structural)
Cell size (Forward Light Scatter)
Cytoplasmic granularity (90 degree
Light Scatter)
Photsynthetic pigments
Extrinsic
Reagents are required
1. Structural
DNA content
DNA base ratios
RNA content
2. Functional
Surface and intracellular
receptors.
DNA synthesis
DNA degradation (apoptosis)
Cytoplasmic Ca++
Gene expression
Flow Cytometry Applications
Immunofluorescence
Cell Cycle Kinetics
Cell Kinetics
Genetics
Molecular Biology
Animal Husbandry (and Human as well)
Microbiology
Biological Oceanography
Parasitology
Bioterrorism
FSC Correlates With Cell Size
SSC Correlates With Internal Complexity
• Fluorochromes Are Molecules That Emit
Fluorescence Upon Excitation With Light
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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)