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

Flow Cytometry

• What is Flow Cytometry?

• 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

What Is It Used For?

DOI 10.1007/s002530100673

• 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

Preparation of flow cytometry

How does it work?

Laser FACS (optional) Data Processing Light Detection Hydrodynamic Focusing Sample Preparation

Step 1: Hydrodynamic Focusing

Step 2: Laser

Step 3: Detection

Step 4 (optional): FACS

Step 5: Data Processing

doi:10.1038/nprot.2009.117

• 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

Questions & Answers

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Advantages

• Measurements on large number of

cells

eg.characterize Ag expression on cell by

cell with large population of cells (important in diagnosis of blood disorders - lymphoma, leukemia.. etc)

• Measured cells can be physically

sorted for further studies

Advantages & Disadvantages

Disadvantages

• Spatial overlap of fluorophores and tedious
• ptimization of experiments
• Non-specific binding of antibodies can make

results difficult to interpret without suitable controls

• Requires a suspension of single cells or other

particles, with minimum clumps and debris.

• > 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)