Cell sorters have become more sophisticated to rival the multicolor capabilities of analytical cytometers with cell sorting experiments becoming more complicated to match. While multicolor sorts are very feasible and can yield excellent results, success is always a product of very careful planning and optimization. From choosing optimal fluorophores to strategies to reducing spillover spreading, this comprehensive article is broken into two parts to give you all you need to play your experiments for advanced accuracy.
Cellular proliferation is a critical component in biological systems. While normal cell proliferation keeps the body functioning, abnormal proliferation (such as in cancer) can be a target for therapy. There are several critical components in developing, validating and optimizing an assay to make these measures using flow cytometry. Knowing the steps to optimize these assays and properly interpret the results will help ensure the best data and best opportunities are pursued.
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.
The most important part of executing a flow cytometry experiment correctly is actually understanding what you are doing. This means you must understand the terms and definitions that are critical to the field of flow cytometry. You must also be able to communicate your methodologies and results intelligently. To this end, we have compiled this list of the top 12 most commonly unknown or commonly misunderstood flow cytometry terms and definitions.
Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. It is also one of the areas that’s steeped in mystery, myths and misinformation. Manually adjusting the compensation values based on how the populations look, or so-called ‘Cowboy Compensation’, is not the correct way to determine proper compensation. The best practices for compensation involve following some very specific rules. Here are 4 steps to correctly compensating 4+ color flow cytometry experiments.
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.
Methods sections in scientific papers are often unable to capture all the critical data necessary to accurately reproduce the results in another lab. Here, information is provided on two specific ways in which flow cytometry researchers are effectively communicating flow cytometry data and metadata to the greater flow cytometry community to improve reproducibility and consistency. These two ways include first, the use of the MIFlowCyt standard and second, sharing data using the Flow Repository.
Reproducibility is a critical component of the scientific process. One cannot publish data if the experiments cannot be replicated. Unfortunately, as Begley and Ellis pointed out in a commentary in Nature that when Amgen attempted to reproduce 53 “landmark” papers in the area of cancer research, only 6 papers were “confirmed.” What does that mean to you, the flow cytometry researcher? To avoid publishing errors that reviews despise, it’s important to follow and promote the best practices in the field, thus ensuring that your data is reproducible to investigators attempting to validate your research. In particular, you must follow these…