Analog instruments processed data differently than the current generation of digital instruments. With analog systems, if the populations were “off-scale,” especially at the low end of the scale, the data accumulated in the first channel. When setting voltage, highly autofluorescent cells would drive the voltage, and it was not uncommon for less autofluorescent cells on the axis to get compressed. Unfortunately, due to the way the data were plotted, this side effect was not always clear to observers.
One question that I get asked on a regular basis is what flow cytometer should I purchase? It’s not as simple as you might imagine. In fact, you need to treat this process as carefully as you would a valuable experiment. There are a lot of variables, and if you’re not asking yourself a huge list of questions, you may miss something critical that will result in the instrument being less than desired. This is a shortlist of questions to ask as you go about the process – your 3-part pocket guide to acquiring the flow cytometer that’s right for…
Experimental validation refers to the process of evaluating a panel and ensuring it’s robust. Sometimes, you're just handed a panel that you have to follow, and it might not be the best panel for your question or even your instrument. There’s a proper way to go about experimental validation, including protocols to follow. Here are 3 parts of high-quality experimental validation.
No matter how advanced or cutting-edge your study is, good science is profoundly dependent on the fundamentals. In fact, no matter your experience level, it is always good to revisit the fundamentals now and again. After all, if flow cytometry were easy, anyone could don a lab coat and get published. In reality, science is as challenging to conduct as it is exciting – that means it’s beneficial for virtually all scientists to take some time and refresh themselves on best practices. You might be surprised to realize what an impact they have on experimental quality…
No researcher wants to discover that the results of a long, careful experiment are confounded by an uncontrolled variable. To assist in data interpretation, you must build careful controls into your experimental workflow. These controls minimize the effects of confounding variables in the experiment while helping to identify the changes related to the independent variable. When designing a flow cytometry experiment, what controls should you consider? Below are a few experimental controls that can dramatically enhance reproducibility in your flow cytometry experiments.
The most common flow assay is undoubtedly immunophenotyping, in which fluorescently tagged antibodies are used to bind to cellular proteins. This allows you to determine the types of cells present. As long as there is a fluorescent reporter available, it is possible to measure biological processes using flow cytometry – especially in a phenotypically defined manner. Probably the most common of these assays is the calcium flux assay. And that is just the tip of the iceberg. In addition to calcium, it is possible to measure magnesium and zinc concentrations, reactive oxygen species, and even membrane potential using flow. Today,…
What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”
In the flow cytometry community, SPADE (Spanning-tree Progression Analysis of Density-normalized Events) is a favored algorithm for dealing with highly multidimensional or otherwise complex datasets. Like tSNE, SPADE extracts information across events in your data unsupervised and presents the result in a unique visual format. Given the growing popularity of this kind of algorithm for dealing with complex datasets, we decided to test the SPADE algorithm in 5 software packages, including Cytobank, FCS Express, FlowJo, R, and the original, free software made available by the author of SPADE. Which was the fastest?
Because mass cytometry allows users to characterize masses so effectively, data can be normalized much more efficiently than what traditional fluorescent flow will permit. If there is no working CyTof at your institution, you can still partner with CyTof-friendly research institutions that have the technology on hand. And because the samples are fixed, you can ship them overnight. This way, they will be analyzed for you. Today’s article will summarize the functionality of mass cytometry technology. This tech has been commercialized largely by Fluidigm in the CyTof systems. There are 5 key points to cover, or takeaways, that cytometrists should…
Reproducibility is key to the scientific method. After the results of a study are published, the community validates the findings and extends them. If the findings are not reproducible, the second step is impossible. With performable experiments increasing in complexity, and the concurrent increase in the cost of equipment and reagents to perform these experiments, it is important to find the best way to maximize the money spent on advancing research. In flow cytometry, there are many places where improvements can be made to increase the consistency and reproducibility of an experiment. The most obvious place is in the instrument,…
All flow cytometer instruments have a certain 3 components, and the way they are put together will dictate the performance of the system. As a user, you’ll be interacting heavily with these components, so you need to know both what they are and how they work. There are fluidics, optics, and electronics. The fluidics allow you to interact at the right flow rate so that your data keep a tight CV. Then you can run the same flow rate for all your samples, and you won't have different CVs for different samples. There are also different optics you can use,…
Did you know that tissues can be measured by flow cytometry? Flow cytometry is the measurement of cellular processes at the whole-cell level. This definition is useful because it includes not only flow cytometry, but any technique that measures at the level of the whole cell. Microscopy, for instance, is a great example of cytometry. But, what can be measured by flow cytometry? For one, tissues with lots of cells. When flow cytometry is practiced, the cells are broken up. Therefore, any cellular interactions within the sample are also broken up. This includes tissues, cell-to-cell contacts in tissues, and virtually…