1 – Instrumentation

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.

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.

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.

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.

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.

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…

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.

When training new users on data analysis, there are several different best practices and gating strategies you should incorporate into your analysis. There are also several misconceptions you must understand. There are 3 gates that many researchers are not using but should be using when analyzing their flow cytometry data. These gates are critical for good data analysis. They will help remove many confounding events that may be clouding your analysis, especially where rare events are concerned.

Forward scatter detectors collect light at small angles relative to the incident beam and can take advantage of the fact that cells preferentially scatter light in this “forward” direction. Forward scattered light is traditionally and often effectively measured with a photodiode, rather than the more sensitive photomultiplier used to measure fluorescence and side scatter. Scatter gets dim very quickly when particles have diameters below the wavelength of illuminating light, considering that scatter intensity decreases with a dependence on r6 of the particle. Here’s how small particles affect light scatter.

The T-Test compares the differences between the means of two populations to determine if the null hypothesis should be rejected. At a minimum, to perform the T-Test, one needs the means and standard deviations of both populations, and the number of measurements. The researcher also needs to set the threshold value, also termed the α. Then, you will compare this threshold to the P-value. If the P-value is greater than the α, there is no significance in the data. However, if the P-value is less than the α, there is significance in the data. Here’s how to run a T-Test.

From instruments, to reagents, to software there are many flow cytometry innovations to pay attention to this year. This article discusses a few highlights of the great things coming your way in 2016 in the field of flow cytometry.

The cell sorting process is inherently stressful. Cells are first manipulated in suspension for up to several hours to prepare and stain them. Then, during the cell sorting process, these cells are pushed through narrow tubing under high pressure in the range of approximately 10-70 PSI, rapidly depressurized after passing through a nozzle, and then jetted through the air at velocities of 20 m/s (~44 MPH) or higher. Keeping cells healthy, happy, vital, and viable over the course of a cell sorting experiment is important to keep cells alive during the sort but also that the recovery of cells from…