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)

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

1. Sterilization equipment2. Balances3. Vortex4. Water purification5. pH meter6. Magnetic stirrer7. Micro pippettor8. Cell counter9. 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 cabinet2. Class II biosafety cabinet3. Class III biosafety cabinet

Vertical laminar flow hoods

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

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

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

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

CO2 and N2 incubators

Inverted Microscope

Fluorescent inverted microscope

Inverted 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: 1. Provides individual measurements of

cell fluorescence and light scattering2. Enables us to individually sort or

separate subpopulations of cells

Fluorescence Activation Process (or Immunofluorescence)

FITC FITC

FITC

FITC

FITC

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. StructuralDNA contentDNA base ratiosRNA content

2. FunctionalSurface and intracellular receptors.DNA synthesisDNA degradation (apoptosis)Cytoplasmic Ca++Gene expression

Intrinsic Extrinsic

Flow Cytometry Applications

ImmunofluorescenceCell Cycle KineticsCell KineticsGeneticsMolecular BiologyAnimal Husbandry (and Human as well)MicrobiologyBiological OceanographyParasitologyBioterrorism

FSC Correlates With Cell SizeSSC 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)

Em

issi

on

S

pect

raE

xcit

ati

on

S

pect

ra

Magnetic-Activated Cell Sorting (MACS)

flow.mp4