Flow Cytometry

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Hanson and I collected samples from 3hr and 6hr OCD-treated rat endothelial cells. These samples were separated into 12 test tubes and brought to the Core Facility in the Medical Science building for flow cytometry testing.

Flow Cytometry is the diagnostic technique used to determine the cell count and their physical characteristics in a fluid medium. It is a powerful technique for the analysis of multiple parameters of individual cells within a heterogeneous population.

Flow cytometers are used in a range of applications, from immunophenotyping, ploidy analysis, cell counting, expression analyses. The flow cytometer performs by passing thousands of cells through a laserbeam and capturing the light the emerges onto the light sensor. The data gather can be converted as digital information to report cellular characteristics, such as size, phenotype, complexity, and health.

Flow Cytometer: The primary system composes of lasers, fluidics system, optics (gather and direct the light), detect (receives the light), computer (converts the signals into digital data and perform the analyses).

Hydrodynamic focusing.The sample travels through the “interrogation point” by one cell at a time flowing through as a single stream. 1-15uM in diameter. As the cell passes through the laser, foward scatter is the amount of light diffracted in the forward fashion that gives information of the cell size. Smaller cells are reported on the left of a histogram, and larger cells are reported toward the right. In side scatter, larger light scattering at different angles is caused by inner granules of the cell. This light scatter is focused through a lense system and focused through a lenses detector. The data must be plotted in a 2D histogram because the cells may be appear to belong in a single population, when in reality they belong to multiple populations that can be discerned by looking the data in a second dimension in a 2D dot scatter plot.

Principle: Fluorescence is the excitation of a fluorophore to a higher energy, resulting in the release of light emission in order to return to its ground state. The state in which the fluorescene is excited is directly related to its emitted light wavelength.

The most common method of flow cytometry involves the use of flourescent labelled antibodies. In these experiments, the labelled antibodies bind to a specific molecule on the cell surface. The light strikes the fluorophore and travels along the a path in which different wavelengths are reflected to their respective light detectors.

Purpose: To distinguish the mode of death of the endothelial cells. Were they apoptotic or necrotic?

Results: We found that 30% of the cells underwent apoptotsis. These endothelial cells in the blood brain barrier may be subjected to programmed cell death in response to ischemic-repurfusion damage.