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Comprehensive Cell Characterization: Techniques, Markers, and Flow Cytometry Insights

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Cell characterization is vital for confirming species origin, assessing tissue correlation, and understanding transformation status. This overview discusses various methods used in cell characterization, including species identification, chromosomal analysis, and the use of tissue and surface markers (e.g., CD11c for dendritic cells, GFAP for astrocytes). Flow cytometry stands out as a robust technique for detecting surface and intracellular factors. The limitations of light scattering in distinguishing cell types and the role of fluorescent dyes for enhanced analysis are also covered, enabling accurate and thorough characterization of cell types.

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Comprehensive Cell Characterization: Techniques, Markers, and Flow Cytometry Insights

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Presentation Transcript

  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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