1 – Instrumentation

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.

How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

By: Tim Bushnell, PhD

The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens. This article describes how to set up, monitor, and maintain optimal voltage settings for your flow cytometry experiment.

How To Troubleshoot The Flow Cytometer Fluidics System

By: Tim Bushnell, PhD

Most of the interactions that a user has with a flow cytometer is with the fluidics system, and many of the issues that users will face in troubleshooting problems on the instrument will also be here. Understanding how the fluidics system works on your flow cytometer will help you prevent many common issues, prepare your samples correctly, and protect your data. Here are four important things to consider about the fluidics system in a flow cytometer.

How To Get A Flow Cytometry Job In 5 Steps

By: Tim Bushnell, PhD

Flow cytometry is a powerful technique impacting both clinical and research. When looking for a career, flow cytometry can take you many places. An experienced flow cytometrist can find a job in a biotechnology company, academia, a clinical setting, and more. To be successful in the field, it’s important to seek out new educational opportunities and network with your peers. Here are 5 tips that can help you turn flow cytometry into a successful career.

Flow Cytometry Procedure For Accurate Sorting Of 5-10 Micron Cells

By: Tim Bushnell, PhD

While cells between 5-10 microns in diameter are typically the simplest cells to sort, quality must still be preserved to prevent sacrificing levels of purity, recovery, and viability. While sorting cells 5-10 microns in diameter does not present a particular challenge compared to other cell types, the standard procedures presented in this article must be followed to guarantee quality sorts, time and time again.

How To Annotate Your Data With FlowJo Keywords

By: Tim Bushnell, PhD

There are numerous different ways to use keywords in FlowJo and other data analysis programs. The problem is most scientists fail to annotate their data properly and pay the price when they want to repeat their experiments. By taking advantage of the keywords listed in this article and by using keyword formulas, you can save time during your analysis. Most importantly, when you go to reanalyze your data, you can utilize your previous keywords and formulas to save even more time.

How Flow Cytometry Optical System Components Work

By: Tim Bushnell, PhD

This article is the second in a two-part series outlining some of the major components of the optical systems used in flow cytometry to provide insight and understanding to what happens before a signal is produced from the PMT detectors. Serving as a knowledge toolkit that can help troubleshoot problems you may encounter when performing your next cytometry experiment, this article investigates lenses, mirrors and filters in your flow cytometry equipment.

What Is A Flow Cytometry Laser And How Flow Cytomtery Optics Function

By: Tim Bushnell, PhD

Understanding the optical system of a flow cytometer may seem unnecessary for performing a typical experiment, but the more you know about your instrument, the better you will be at understanding your data, as well as troubleshooting potential issues. This article breaks down 4 elements of flow cytometer optics to provide a broad understanding on its impact on fluorescence.

What Is Fluorescent Activated Cell Sorting And 4 Other Questions About FACS Data Analysis

By: Tim Bushnell, PhD

The last 40 years have seen significant advancements in cell sorting technology. Cell sorting is often the entry point for many experiments. Fluorescent Activated Cell Sorting (FACS) combines the traditional power of flow cytometry and couples it with the ability to isolate the cells of interest. Understanding the inner workings of the instruments and some rules for preparing samples will lead to more successful experiments. Here are 4 essential facts about FACS.

How To Create A Flow Cytometry Quality Assurance Protocol For Your Lab

By: Tim Bushnell, PhD

Implementing a system of quality assurance protocols lends confidence to the data collected, especially for those researchers performing longitudinal studies. Optimal instrument setting, cytometer sensitivity, and monitoring of day-to-day variability in measurement leads to improved assurance for those using this instrument to collect their critical data. QC programs will continue to be prudent measures for cytometrists to take as they align with the current emphasis on quality and reproducibility.

4 Spectral Viewers You Should Be Using For Your Flow Cytometry Experiments

By: Tim Bushnell, PhD

Fluorochrome emission is the lifeblood of flow cytometry. The use of in silico tools, like spectral viewers, can save a lot of effort and missed opportunity by allowing for the modeling of excitation and emission profiles in the context of what filters a given instrument is equipped with. Using these tools, it is easy to identify where a new fluorochrome will be measured on an instrument, where a fluorochrome may cause issues with other fluorochromes, and what filters are best for detection. These tools can save a lot of troubleshooting at the beginning of an experiment, and also help provide…

The Difference Between Purity, Single Cell, And Recovery Cell Sorting Techniques

By: Tim Bushnell, PhD

Sorting efficiency, in fundamental terms, is a real-time measurement, generated by the instrument, of how successfully its sorting system is able to resolve cells that we want to sort (target events) from cells we do not want to sort (non-target events). In order for the instrument’s sort output be acceptable with respect to the researcher’s needs, it is not sufficient to simply tell the instrument WHAT to sort, but is also critical to tell the instrument HOW to sort the target population. The HOW is determined by the sort modes.