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Characterizing Cells

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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Characterizing Cells

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  1. Characterizing Cells

  2. 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)

  3. 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

  4. 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

  5. Principles Of Flow Cytometry

  6. How Is It Done: http://biology.berkeley.edu/crl/flow_cytometry_basic.html

  7. Limitations With Light Scattering • 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

  8. FSC vs SSC Dot Plot

  9. Fluorescence And Antibodies To The Rescue

  10. Fluorescent Dyes And Antibodies • 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)

  11. Principles Of Fluorescence E= h f  = f 

  12. Excitation Spectra Of Fluorochromes

  13. Emission Spectra

  14. Architecture Of A FacsCalibur Instrument

  15. Analyzed Data

  16. GM-CSF+IL-4 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

  17. Unstained ISOTYPES

  18. GM-CSF + IL-4 (6 Days Old) GM-CSF

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