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Flow Sorting
What is Flow Sorting?
Sorting can be defined as the physical separation of a cell or particle of interest from a heterogeneous population.
How Does Sorting Work?
In general, flow cytometers use a principle involving the electrostatic deflection of charged droplets similar to that used in ink-jet printers. Cells are aspirated from a sample and ejected one by one from a nozzle in a stream of sheath fluid which is generally PBS but can be any ionised fluid.
All streams are unstable with respect to time and will eventually break up into droplets. It is possible to stabilise this break-off point by applying a stationary wave of vibration of known frequency and amplitude to the stream.
As the cell intercepts with the laser beam, scattered light and fluorescence signals are generated and the sort logic boards make a decision as to whether the cell is to be sorted or not (according to user-defined criteria).
The distance between the laser intercept and the break-off point is called the drop delay. If a cell of interest, i.e., one to be sorted, has been detected, the cytometer waits until that cells has travelled from the intercept to the break-off point and then charges the stream.
So as the drop containing the cell of interest leaves the solid fluid stream it will carry a charge, either positive or negative. A further distance downstream the charged drop passes through two high voltage deflection plates and will be attracted to towards the plate of opposite polarity.
So it is possible to sort two separate populations from the same sample. By applying two different levels of charge to the left or the right streams, it is actually possible to sort two streams either side, and both the Cytomation MoFlo and the Becton Dickinson DiVa are capable of this.
The theory and the practicalities of cell sorting are rather more complicated than this simple overview. However, it is also useful to know that we can alter the mode of sorting to give maximum purity, maximum recovery (if a small, precious population is required) or maximum count accuracy (for cloning).
In the UK, the Cytometry section of the Royal Microscopical Society organises periodic courses teaching the theory and practice of cell sorting.
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