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.
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.
Good flow cytometry depends on a high quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze. Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Sample preparation becomes the critical first step in any flow cytometry experiment. To get high quality results, follow these 3 sample preparation steps.
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.
Every experiment has the goal of ensuring consistent and reproducible data. This makes the proper use of controls to establish the boundaries of gates critical. With the exception of one controversial control discussed in this article, each one of these gating controls plays an important and specific role toward the goal of reproducibility. Using these gating controls in every experiment will reduce data variability within the experiment, as well as between labs and institutions.
Flow cytometry (FCM) datasets that are currently being generated will be two orders of magnitude larger than any that exist today. Reproducibility continues to be a critical area that all researchers need to be aware of and researchers need to keep up on best practices to stay relevant. One area that flow cytometry researchers should be focusing on is the emerging changes in the area of automated data analysis. This brief article explains why.
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.
Multicolor sorting experiments can be complicated and if not setup properly, result in wasted time and suboptimal results. When setting up a multicolor experiment, the most saliently critical step is to set PMT voltages properly. In addition, using a viability dye and addressing doublet discrimination and setting the right sort regions and gates is important for any kind of flow cytometry experiment, but particularly for cell sorting. These tips help to ensure your setup is perfect to achieve results of the highest caliber.
When sitting down to perform a new analysis of flow cytometry data, the researcher is guided by very particular laws of nature and a very specific method of working through a biological hypothesis to avoid shaping the results to his or her whims. Following these 5 data analysis and gating strategies through the hierarchy described in this article, researchers are provided with several strategies for identifying and displaying the most relevant data from their flow cytometry experiments.
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.
When preparing figures for publication, the scientific question and hypothesis that forms the basis of the paper must be central and all the figures must be in support of that. The flow cytometry data that forms the basis of the conclusions should be presented clearly and concisely. While it provides pretty pictures and colorful layouts, the meat of the data are the numbers ― percentages of populations, fluorescent intensity levels and the like ― are what will convince the reader that the hypothesis tested is valid and well thought out. Here’s how to choose the correct flow figure for presenting…