Cell sorting remains the best tool to isolate and purify cellular populations that can be phenotypically defined. This is especially true for rare-event detection and purification. Successful rare event detection and purification requires some attention to ensure the best yield and purity.
1) Watch the instrument to ensure success
a) Keep the core stream tight – the tighter the core stream (low differential pressure), the tighter the CV on resulting data, therefore the easier it is to determine best placement of gates for sorting. Tight core streams also reduce coincident events (two cells passing through the intercept at the same time). Since flow cytometry requires single cells for proper analysis, the
more doublets, the fewer sorted cells.
b) Eliminate the aborts – aborts arise from a cell arriving at the laser intercept while the electronics are processing the previous pulse. This new event is lost (aborted). This results in loss of cells as well as decreased purity. Optimizing the window extension for the cell type can have a dramatic effect in improving the quality of the sort (and reducing the abort rate).
2) Avoid the artifacts
a) Viability dyes – it cannot be stressed enough that a simple viability dye improves the quality of the sort for downstream applications by reducing the mis-sorted false-positive cells that are dead.
b) Avoid the aggregates – in establishing a gating strategy, consider adding a pulse gate (height vs area, for example) to eliminate cells that are aggregated.
c) Gate for success – using the appropriate controls for gate placement and avoid the use of the histogram. Histograms mask data and make it impossible to find rare events. Take a look at this data, courtesy of Jennifer Wilshire, for measuring GFP fluorescence. Looking for the GFP population
with a histogram is very difficult. Plotting a bi-variate plot (against a PE signal, in this case) helps identify the cells that are truly GFP positive (bottom right). It also assists in demonstrating that the dim positive GFP cells can be separated from autofluorescence.
d) Threshold low – when a threshold is setup, the cytometer is blinded to any signal that is below that threshold value. So while it is possible to eliminate debris and such from the data using a high threshold, if the cell sorter cannot see the debris, it will find its way into the sort tube and contaminate the cells for downstream applications.
So, when setting up a gating strategy on a cell sorter – either with an operator or alone, make sure the steps above have been addressed. This will help ensure a good sort, even for the rare populations!
ABOUT TIM BUSHNELL, PHD
Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.More Written by Tim Bushnell, PhD