Cytometry Lab

Introduction to Flow Cytometry

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Flow cytometry is the measurement (meter) of characteristics of single cells (cyto) suspended in a flowing saline stream. A focussed beam of laser light illuminates each moving cell and light is scattered in all directions. Detectors placed forward of the intersection point or side-on (with respect to the laser beam) receive the pulses of scattered light and they are converted into a form suitable for computer analysis and interpretation. The total amount of forward scattered light detected depends on cell size and refractive index but is closely correlated with cross-sectional area of the cell as seen by the laser, whereas the amount of side scattered light can indicate nuclear shape or cellular granularity.

Further properties of the cell, such as surface molecules or intracellular constituents, can also be accurately quantitated if the cellular marker of interest can be labeled with a fluorescent dye; for example, an antibody-fluorescent dye conjugate may be used to attach to specific surface or intracellular receptors. Other dyes have been developed which bind to particular structures (e.g. DNA, mitochondria) or are sensitive to the local chemistry (e.g. Ca++ concentration, pH, etc.).

It is an obvious requirement that the laser beam in use is of a suitable colour to excite the fluorochrome or fluorochromes used. The quantity of fluorescent light emitted can be correlated with the expression of the cellular marker in question. Each flow cytometer is usually able to detect many different fluorochromes simultaneously, depending on its configuration. In WEHI instruments, between 3 and 15 fluorochromes may be analysed simultaneously. To achieve this, in a number of WEHI's cytometers two three or four separate laser beams of different colours are used so that this wide range of fluorochromes may be simultaneously excited. For more information about choosing an appropriate set of fluorochromes, see Fluorescence in Cytometry.

The WEHI Cytometry laboratory has FACScans, FACSCaliburs and LSR cytometers (Becton Dickinson) available for three colour or up to 11 colour analysis.

A further five instruments (Becton Dickinson and Dako) are capable of cell-sorting as well as analysis.

Cell Sorting:

Cells can be selected for sorting based on all of the measured properties of fluorescence and light scatter. The cell suspension is injected into the centre of a flowing saline stream within a tapered nozzle. This saline stream that carries the cells, begins as a continuous stream as it is projected from a circular hole (typically 70um diameter) in the nozzle tip. The laser beam illuminates each cell just after it emerges from the nozzle. Data is collected at this point (known as the point of analysis) and a decision is made as to which cells are to be collected.

At the same time, a high frequency vibration continuously applied to the nozzle assembly causes the stream to break up into droplets at some distance down from the tip. This distance is remarkably stable and repeatable for all droplets breaking off. Therefore, having accurately determined the time elapsed from the point of analysis to when each droplet breaks from the stream, it is possible to set the sorter so that, whenever a cell is analysed and is found to fit our predetermined sort criteria, it waits until the cell is about to enter the droplet breaking off from the stream, then applies an electrical charge to the stream and momentarily holds it on until the droplet breaks off. The stream then loses the charge but the detached droplet retains charge and is deflected by charged metal plates sitting on either side of the stream. The droplet is deposited into a collection vessel.

The cells collected are essentially unharmed by the process. They can be measured subsequently for in vitro or in vivo responses or analyzed at the RNA or DNA level.

Where many fluorochromes must be excited, up to 4 laser beams may be used to sequentially illuminate the cell as it travels down the sheath saline stream.

The cells may be sorted into tubes where large numbers are required (as in the figure) or small, accurately counted numbers or even individual cells may be deposited directly into microtitre plates.

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