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Characterizing Cells. What To Characterize. Confirmation Of Species Of Origin Correlation With The Tissue Of Origin Transformation Status Finite Or Continuous Cross-Contamination Stability (ex. susceptibility to transformation). How Is Characterization Done. Species Identification

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What to characterize
What To Characterize

  • Confirmation Of Species Of Origin

  • Correlation With The Tissue Of Origin

  • Transformation Status

  • Finite Or Continuous

  • Cross-Contamination

  • Stability (ex. susceptibility to transformation)


How Is Characterization Done

  • Species Identification

    • Chromosomal analysis

  • Tissue Markers

    • Cell surface markers

      • Ex. CD11c if DC

    • Intermediate filament proteins

      • Ex. 1 Glial fibrillary acidic protein (GFAP) for astrocytes. Ex. 2 Desmin for muscle cells. Ex. 3 Cytokeratin for epithelial cells

    • Differentiated products

      • Ex. Melanin for melanocytes, hemoglogin for erythroid cells, serum albumin for hepatocytes

    • Unique Markers

      • Ex. HLA highly polymorphic, unique to an individual

  • Morphology

    • Easy and fast but has variability depending on culturing conditions and site


Flow cytometry is a powerful technique for characterizing cells
Flow Cytometry Is A Powerful Technique For Characterizing Cells

  • Allows For Detection Of Surface Markers Of Cells

  • Allows For Detection Of Intracellular Factors

  • Allows Detection Of Secreted Factors By Cells

  • Allows For Detection Of DNA Content



How is it done
How Is It Done: Cells

http://biology.berkeley.edu/crl/flow_cytometry_basic.html


Limitations with light scattering
Limitations With Light Scattering Cells

  • Some Information Can Be Obtained

  • FSC Correlates With Cell Size

  • SSC Correlates With Internal Complexity

  • To Distinguish Between 2 Cell types

    • A. Size Has To Be Different OR

    • B. Internal Complexity i.e amount of granules

  • If These Two Parameters Are The Same, Then No Distinction Can Be Made

  • See The Following Figure



Fluorescence and antibodies to the rescue
Fluorescence And Antibodies Cells To The Rescue


Fluorescent dyes and antibodies
Fluorescent Dyes And Antibodies Cells

  • Fluorochromes Are Molecules That Emit Fluorescence Upon Excitation With Light

    • Ex. 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)


Principles of fluorescence
Principles Of Fluorescence Cells

E= h f

 = f 






GM-CSF+IL-4 Cells

GM-CSF+IL-4+PGE2

Fig. 2

GMF: 1.85

GMF: 168.92

25

75

99

1

GMF: 11.82

GMF: 391.83

99

80

1

20


Unstained Cells

ISOTYPES



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