Sorting

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

Sorting 2

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!

Join Expert Cytometry's Mastery Class
Tim Bushnell, PhD
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.

Similar Articles

Getting A New Flow Cytometer? Try Before You Buy (And 2 Other Tips)

Getting A New Flow Cytometer? Try Before You Buy (And 2 Other Tips)

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

5-Point Guide To Buying A New Microscope For Your Lab

5-Point Guide To Buying A New Microscope For Your Lab

By: Heather Brown-Harding, PhD

Have you ever noticed how painful it can be to purchase a new microscope? It would be hard to miss – this can be a frustrating process. A lot of scientists and students consider the new microscope hunt quite scary for a variety of reasons. It might be that you’re worried you won’t get the right microscope and that you’ll regret it, or you may find that dealing with salespeople, in general, makes you kind of uncomfortable. But remember, salespeople are just human beings like you and me, and if we can treat them as such, the whole process of…

Ask These 7 Questions Before Purchasing A Flow Cytometer

Ask These 7 Questions Before Purchasing A Flow Cytometer

By: Tim Bushnell, PhD

I am still convinced that my first cell sorter was possessed. The number of issues that I had with the system remains hard for me to believe, even after all these years. It had been purchased, in part, from one vendor because the sales rep for a competitor was nowhere to be found. At that time, I admit I wasn’t overly diligent in my research process. Since then, I’ve pinpointed some critical questions that need to be answered before purchasing a new instrument. At the end of the process, a shiny new instrument will arrive at your facility. Make sure…

Instrument Quality Control For Reproducible Flow Cytometry Experiments

Instrument Quality Control For Reproducible Flow Cytometry Experiments

By: Tim Bushnell, PhD

The flow cytometer is an integral component of any flow cytometry experiment, and special attention should be paid to ensuring that it is working correctly and consistently. As an end-user, the researcher should be able to sit down at a machine and know that it is performing the same way today as it was yesterday and last week. Equally important is that if any changes in instrument performance have occured, the end-user knows how they have been addressed and corrected, rather than letting them fester and potentially affect the results. Quality control measurements can include a variety of targets, such…

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

By: Tim Bushnell, PhD

Preparing for rare event analysis requires an understanding of the power and limitation of the instrument to be used. From how fast to run the fluidics, to how the signal is processed to the number of gates that can be used in the sorting experiment, each factor impacts the outcome of the experiment.

3 Ways The ZE5 Cell Analyzer Accelerates Flow Cytometry Research Opportunities

3 Ways The ZE5 Cell Analyzer Accelerates Flow Cytometry Research Opportunities

By: Tim Bushnell, PhD

Some technological advances are incremental, while others are significant game-changing tools that offer the researcher the ability to significantly improve current assays while allowing for new and novel avenues of research to be performed. With speed, sensitivity, and capacity to spare, the ZE5 fits into the game-changing category. Reduced carryover, increased speed of acquisition, and a large number of parameters all open up new and novel assays while improving the quality and reproducibility of ongoing ones.

3 Advantages Of Using The ZE5 Cell Analyzer

3 Advantages Of Using The ZE5 Cell Analyzer

By: Tim Bushnell, PhD

Since the first laser was mounted to create the first flow cytometer, there has been a push for more - more lasers, more detectors, more colors. As a result, today’s researchers require a large number of lasers and detectors to ensure current panels can be run and new, expanded panels can be developed. This can be problematic because, in general, making one decision to improve a cell analyzer can limit the analyzer in other ways. It may seem like an impossible task, but the team of Bio-Rad and Propel Laboratories, collaborated to bring the ZE5™ Cell Analyzer to the market…

3 Advantages FCS Express 6 Has Over Other Flow Cytometry Data Analysis Software Programs

3 Advantages FCS Express 6 Has Over Other Flow Cytometry Data Analysis Software Programs

By: Tim Bushnell, PhD

FCS Express is the ideal data analysis software program to use when analyzing your flow cytometry experiments because it is the most user-friendly program available that is both aligned with current data analysis best practices and maintains rigorous quality control standards.

How To Use A Threshold To Reduce Background Noise In Flow Cytometry

How To Use A Threshold To Reduce Background Noise In Flow Cytometry

By: Tim Bushnell, PhD

Getting a clear signal with reduced noise is an essential component to good data. Adding a threshold when acquiring flow cytometry data is one way to do that. It reduces the number of events by setting a bar that a signal pulse must clear before it is counted as an event. Depending on the importance of the data, the downstream applications for the data (or sorted cells) will dictate how critical the threshold is. In combination with proper sample preparation, appropriate thresholding will reduce debris and ensure best outcome.

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.