Flowing Software 2.5 1

Flow Cytometry is everywhere today. Easy free ways to view flow cytometry FCS files are not. There are many great commercially available software applications that allow not only for data visualization but also advanced statistical analysis of the data. Unfortunately, they cost hundreds to thousands of dollars. Most of the time the price is so big that they don't even put the quote on their website. Even with a paid license, there are usually limits placed on the number or location of computers that the software can be used on. This is acceptable for large labs and big companies, but smaller groups who are more limited on resources may not be able to use them.

Given the importance of flow cytometry to so many different scientific fields in today's world, analysis software that is usable by all should exist. There have been several projects that have sought out to create a free software analysis package for FCS files. Some are better than others. We are here to go through the biggest and most complete free flow cytometry software available and discuss their pros and cons.

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There are a couple of other places to find reviews of free flow cytometry software. Samani et. al. (2014) took a look a 4 different free software applications in a paper you can find here. Even though this paper is several years old at this point, most of the software they reviewed has not received any updates since they published this, so most of their points still apply.

Another often-cited resource for free flow cytometry software information is J. Paul Robinson's software catalog over at Purdue University Cytometry Laboratories. The page gives a good overview of many different types of resources. It has a lot of great software on it that we have found really useful. It also contains a lot of software that is either really old and meant to be used with old file formats and data, or software that is very specialized to do a specific task. However, it is missing a few newer software packages.

We were programmers long before we did flow cytometry. We will give a brief, high-level description of the important software design decisions that were made by the software programmers and discuss the trades offs of those decisions in general terms.

We will take a look at what it takes to get started using the software. What dependencies does it have that the user needs to set up? How much personal information does the user need to provide? Is the software web-based or does it require you to download executables?

There are a lot of features that are pretty standard that all flow cytometry analysis software should be able to perform. Then there are some nice to have features that are helpful in a lot of situations. Finally, there are some very specific features that most people don't really use at all. We see how each of the programs stack up.

One of the major purposes of flow cytometry analysis software is to visualize data. So, how nice does the data look? We will open up a test file in each program and give you screenshots of dot plots and histograms. The images displayed will be the default view that each program gives for a dot plot and histogram.

There can be a lot of computation that is involved when loading FCS files and calculating gates and statistics. The speed at which this is done depends a lot on the software design decisions of the programmer. It depends on how big your usual FCS file is, how many you have open, and how many gates and stats you apply. If the program calculates inefficiently, the noticeable lag can be quite detrimental to the user experience. We will benchmark the different programs doing a few common tasks to get some estimate of their performance.

One of our motivations for starting Floreada.io is that existing software was not compatible with our computers and we wanted a cross-platform app. Therefore we decided on a web-based program that would work on any device with a web browser, including Windows, Mac, Linux, iOS, and Android. Although code that runs in a web browser will be slower than equivalent native code, we thought the tradeoff was worth it.

There are two general ways to run code on the web - the server-side (our computers) or the client-side (your computer). All existing flow cytometry analysis software that we could find uses a primarily server-side approach. Basically, all your data gets transferred to the server and when you add a gate or statistic or what to draw a dot plot, your computer will send a message to the server and ask it for the image data, or statistical information. The server will do any calculations and send your computer back the information it needs. This approach makes the already slow web-based approach even slower.

Regardless, we are constantly looking for ways to improve performance and have a few upgrades we can make that will likely put Floreda.io on par with any native based flow cytometry software. Stay tuned.

FCSalyzer still seems to be in somewhat active development. It was initially published in 2012 and has received multiple releases every year since then until 2019. Although it hasn't yet been updated in 2020, the developer is active on the programs SourceForge page and seems to be really active in helping users and tracking down bugs. The developer has done a great job and has been going strong for almost a decade at this point. FCSalyzer is one of the only free flow cytometry analysis software projects that remains active.

Flowing Software is a free analysis program that is no longer currently in development with no releases since 2013. It was created by the same person who made Cyflogic and shares many visual characteristics. It's much better software than Cyflogic though. It is available for Windows computers only.

WinMDI only needs a summary. You almost certainly should not be using it. It is an extremely old 16 bit Windows program that was made before the FCS3.0 standard. It may have worked well decades ago but it is completely obsolete at this point. It probably won't even run on your computer. The only situation where it might be useful is if you have extremely old computers in your lab. Other than that find something more modern. The only reason we have it on this list is that it is often on other lists of free flow cytometry software (usually lists that haven't been updated in a while).

Flow Jo offers a 30-day free trial for you to try. They are very protective of their software and will require you to input hardware identification before they send you a temporary key. You can find the free trial here

Beckman Coulter offers a free trial of its flow cytometry software, Kaluza, for 30 days. Again, you will need to fill out a bunch of forms to get access. You can start the process here on their website.

Flowlogic Software from Miltenyi Biotec also has a free trial version of their analysis software available for 30 days. They have the biggest forms of all to fill out. Head over to their website here to start signing your life away.

A lot of people, especially students, don't have the resources to afford an extremely expensive license or subscription to professional level flow cytometry analysis software. Without paying there are essentially two options: using a free trial of commercial software or using freely available software. Although many small free flow cytometry analysis software projects have been started, most have ceased active development many years or even decades ago. Floreada.io was started to fill this gap and create easy to use free analysis software that is accessible on all platforms. We hope you will give us a try - it's super easy to get started. Given how important flow cytometry has become to so many different scientific fields, there should be an easy free way to do simple analysis that everyone has access to. Come help us accomplish this goal. Get started here

If you are one of the authors of software we have listed above, feel free to contact us if you want anything added or removed from what we said. If you have a free flow cytometry program that we didn't list here and you want to be added, let us know.

To elaborately analyze graphs, these software also provide tools like create region, marker, quadrants, zoom, pan, etc. These let you save statistical data to a TXT file. You can even export plot to an image like PNG, BMP, etc.

Flowing Software is a simple and easy to use flow cytometry software for Windows. It comes with various tools to analyze data from flow cytometry experiments. It lets you perform flow cytometry analysis on data stored in FCS and TXT (tab delimited) files. It provides a wide range of tools in different categories which are as follows:

FlowPy is the next free flowing software for Windows in this list. You can open a FCS file or a Text file and then choose a plot type for flow cytometry analysis. It provides Histogram, Dotplot, and Cumulative Distribution graphs to visualize flow cytometry data. You can choose a parameter to plot respective graph, such as FSC-A, FSC-H, FSC-W, SSC-A, SSC-H, SSC-W, Time, etc. Checkout this link to know about these parameters.

Cyflogic is another free flowing software to analyze flow cytometry data. It provides Dot Plot, Histogram, Histogram Modeller, Overlay Histogram, 3D Plot, and ClipBook statistical tools to visualize FCS files. There are a lot of features provided in this software to let you perform flow cytometry analysis. These include following options:

Methods: A Beckman Coulter Epics XL flow cytometer with argon ion laser operating at 488 nm was used for flow cytometry analysis. The voltage and the gain settings for individual channels were set at high voltage and gain for the detections of autofluorescence, fluorescence of adsorbed Congo red, forward scattering (FSC) and side scattering (SSC) intensities from the aggregates of proteins and nanoparticles. Each sample was analyzed to characterize and quantify the number of aggregates with a limit of maximum 20,000 events. The flow cytometry data were analyzed using Flowing software version 2.5.1 and Origin 8.0.

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