Flow Cytometry Techniques Caf Andrew Sharon Jack HyungJo Hwang - - PowerPoint PPT Presentation

Flow Cytometry

Techniques Café

Andrew Sharon Jack HyungJo Hwang Justin Liang Mizuki Kurashina

• High-throughput technique to

analyse and/or sort cells

• Utilizes microfluidics and lasers to

measure parameters

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

○ Light source (mercury lamp or laser) ○ Flow chamber ○ Optical filter ○ Photomultiplier for detection of wavelengths ○ Data processing unit

• 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

• Hydrodynamic focusing allows

single cell to move through the laser beam

Step 2: Laser

• Forward scatter (FS) proportional

to size of cell detected

• Side scatter (SS) proportional to

granularity of cell

• Detect presence of fluorophore

via wavelength

Step 3: Detection

• Each cell moves through laser and

is individually analyzed

• FS and SS light, and fluorescence

are split into defined wavelengths

• Optical filter filters light so that

each sensor detects fluorescence at a specific wavelength

Step 4 (optional): FACS

• As cells pass through the laser, the cell is

given an electronic charge, depending on the fluorescence of the cell

• Deflection plates attract cells accordingly

into collection tubes

• Advantageous because you can purify two
• r more populations for further research

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)