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Flow Cytometry What is Flow Cytometry? What Is It Used For? - PowerPoint PPT Presentation

Flow Cytometry What is Flow Cytometry? What Is It Used For? Measure many different parameters on the same cell Protein expression DNA content Immunophenotype cells Sorting cells into different tubes based


  1. Flow Cytometry

  2. What is Flow Cytometry? ● ●

  3. What Is It Used For? Measure many different parameters on the ● same cell Protein expression ○ ○ DNA content Immunophenotype cells ● ● Sorting cells into different tubes based on phenotype (Fluorescence-Activated Cell Sorting: FACS) Determine what phase of the cell cycle a ● cell is in DOI 10.1007/s002530100673

  4. Preparation of flow cytometry Requires 5 operating units ● ○ ○ ○ ○ ○ Many cells (100,000+) ● ● Determination of what will be measured (Size, Granularity, Expression of Protein, etc.) Necessary reagents for different conditions ● ○ Size and Granularity: no reagents required Expression of Surface Proteins: Antibodies with conjugated fluorophore against ○ protein of interest, additional secondary antibodies against primary antibodies ○ Expression of Cytoplasmic Proteins: Fixation and pemeabilization of cell (ex. Paraformaldehyde/Saraponin) ○ Expression of Secreted Proteins: Golgi block

  5. How does it work? Sample Data Preparation Processing Hydrodynamic FACS Focusing (optional) Light Laser Detection

  6. Step 1: Hydrodynamic Focusing ●

  7. Step 2: Laser ● ● ●

  8. Step 3: Detection ● ● ●

  9. Step 4 (optional): FACS ● ● ●

  10. Step 5: Data Processing ● The amount a cell scatters or fluoresces light is measured and displayed as a histogram eg. allows us to detect abnormal imbalance in CD4+/CD8+ T cells -> to see if HIV has resulted in actual immuno-suppression doi:10.1038/nprot.2009.117

  11. Questions & Answers Thank you for watching our presentation!

  12. Advantages & Disadvantages Advantages Disadvantages Measurements on large number of - Spatial overlap of fluorophores and tedious - optimization of experiments cells eg. characterize Ag expression on cell by - Non-specific binding of antibodies can make cell with large population of cells results difficult to interpret without suitable (important in diagnosis of blood controls disorders - lymphoma, leukemia.. etc) - Requires a suspension of single cells or other - Measured cells can be physically particles, with minimum clumps and debris. sorted for further studies -> This means that the tissue architecture and any information about the spatial relationship between different cells are lost when single cells or nuclei are prepared. -> does not work well for cells that tend to stick together (eg. carcinoma or sarcoma)

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