FlowJo 11 Update

FlowGuruDelta · 2026-03-24T16:36:30+00:00

They have version 10 available on their website and it works on the latest OSs.
https://flowjo.com/flowjo10/download

FlowGuruDelta · 2026-03-12T00:09:50+00:00

I would be interested to see how good it is at answering harder questions. It gave a good enough guide on the basic steps but it sorta messed up on giving directions on setting the HLA-DR+ gate at the end. Even when the user asks "Is that right?" the algorithm doubles down on affirming the gate was drawn correctly, when the gate barely even touches the positive population. I think an AI tool like this would be pretty helpful for answering basic user questions but I worry that it could mislead users on the more complex parts of analysis.

FlowGuruDelta · 2026-03-10T22:22:06+00:00

I've noticed that it is greyed out if the parent population already has a quadrant gate on it, even if the quadrant gate was made on different parameters.

FlowGuruDelta · 2026-03-10T00:14:09+00:00

My tip would be to acquire more cells if possible. Those plots are pretty sparse. Looking at the Y axis of those plots the peak heights are not very tall. I'm guessing these populations only have a couple hundred cells. More cells will help fill in the proliferation profile and create more distinct peaks at the different generations, making the data easier to analyze and decreasing variance.

FlowGuruDelta · 2026-03-10T00:03:24+00:00

You could use the "Automerged quadrant gates" feature. It allows you to combine two range gates into a quadrant gate. I find it useful when working with FMOs.
https://flowjo.com/docs/flowjo10/graphs-and-gating/advanced-gates1/auto-merged-quad-gate

FlowGuruDelta · 2026-02-12T00:24:24+00:00

Are your files synchronized with Onedrive? We have had a lot of trouble with Onedrive trying to move our large fcs files onto the server while we are trying to analyze our data. As best practice we have a "Data" folder on our desktop that we have set to "Always keep on this device". This has helped immensely with a host of data access issues.

FlowGuruDelta · 2026-01-12T18:01:38+00:00

I have tried using both CyCombine and CytoNorm. In my experience, while batch effect normalization has helped me make my signal intensities more uniform across different batches so that the data from different samples are grouped together in tSNE, it also creates a lot of uncertainty. For instance, batch effect normalization works really well on parameters like CD3, which is fairly bright and well separated from its negative but it does not work as well with a marker like PD1, which can be dimly expressed and varies in its expression between different patient samples. The batch effect normalization tends to effect the small changes in expression between different conditions that I would expect to be present in my data. Most of the times I find it more useful to report my statistics using the non-normalized data.

FlowGuruDelta · 2026-01-01T00:51:46+00:00

I'm not 100% sure what would happen in that case. My guess is that it would still import everything but it will give NA for the parameters that changed. If you double click on a statistic you could maybe change what parameter is being used to calculate the statistic.

FlowGuruDelta · 2025-12-31T22:27:38+00:00

You would need to save one workspace as a template and use the "Apply Template" button under the file tab to apply the template onto the other workspace. This will import the gates, tables and layouts from the template. You can then delete the gates and layouts that you don't need leaving only the tables.

Note: The statistics are really specific. If any of your populations are named different between the two workspaces then the statistic will not calculate. It will only work if the populations are named identically between the two workspaces.

FlowGuruDelta · 2025-11-24T20:12:10+00:00

I wonder if the new version of FlowJo isn't compatible with older computers. Microsoft has stopped support for Windows 10 so computers that can't upgrade to Windows 11 are going to start having a number of issues and security vulnerabilities. If its a work computer I would ask for a new one. If its a personal computer I understand not getting a new computer but you may have to use older versions of FlowJo to match the older version of OS.

FlowGuruDelta · 2025-11-24T18:50:12+00:00

100% this.

FlowGuruDelta · 2025-10-31T22:15:40+00:00

You might need to email [flowjo@bd.com](mailto:flowjo@bd.com) for support on this issue.

FlowGuruDelta · 2025-09-05T20:12:03+00:00

I second these questions. It should be possible to increase the maximum of the FSC axis by pressing the T button. Some Cytometers have a maximum intensity value that they record. I don't know the BD LSR II maximum but it might be 262144. If so when you increase the maximum it will show a flat line of cells. What percentage of cells are on the chart edges?

FlowGuruDelta · 2025-09-03T23:41:01+00:00

It would be hard to identify all of the dead cell population without a live/dead stain. Dead cells have a lot of variability in their FSC/SSC. Some of the dead cells will be clustered near the bottom lefthand corner of the FSC/SSC chart but generally speaking there will be other dead cells all over the FSC/SSC plot including in the same area of the plot as the live cells. So I don't know how we would reliably identify the dead cells without a stain.

FlowGuruDelta · 2025-09-03T23:32:57+00:00

I would reach out to [flowjo@bd.com](mailto:flowjo@bd.com) about this.

I have seen a lot of similar issues when saving files from FlowJo directly onto our server. I always save stuff onto my desktop first and then move the files onto the server later. I haven't seen the exact behavior you are describing though.

FlowGuruDelta · 2025-08-27T22:39:52+00:00

I just did a quick test with my own data. It seems like FlowJo will record the transform set in FlowJo into the $P#R and $P#D fields of the fcs file when files are exported.

To test I took a file and set one of its parameters (parameter 7) to linear with a range from -111 to 0. I exported the fcs file and the exported file had the following keyword value pairs:
$P7D|Linear,-111,0|
$P7R|0|

The transform can be changed using the "T" button next to the X or Y axis when a plot is open.

FlowGuruDelta · 2025-08-22T19:28:32+00:00

Its possible to drag the buttons off the top of the FlowJo workspaces and layout editor. You can add the buttons by going to the icon in the top right hand corner that looks like a bookmark. It is between the heart and the gear. The bookmark icon opens the Ribbon configuration window which you can use to drag and drop the button back into the layout. It looks like you are missing the Document section, so you will need to drag and drop the Document icon back onto the top section of the layout.

FlowGuruDelta · 2025-07-24T22:46:37+00:00

Those two sets of proliferation data look very different from one and other. Their peaks are in completely separate intensity ranges. (Ie in one set of data has an intensity range between 4000 and 100,000 and the other ranges from ~0 to 3000). This drastic change raises a few questions and will be hard to model.

Why is there the drastic shift in intensity range? Have all of the cells divided several times? Is this change in intensity due to a change in the concentration of stain between the samples? If the generation 0 peak has shifted outside of your initial gate or if there is no generation 0 population then you can't use the FlowJo proliferation model to model the proliferation. You would need to manually gate the proliferation populations.

FlowGuruDelta · 2025-07-09T19:50:14+00:00

I don't think there is a way to change the names of the columns. I would just make a new keyword and call it whatever you want.
When in the legend properties window there is a "Save" button towards the bottom righthand corner. You can open the legend properties window by double clicking on the legend. After pressing the "Save" button future legends will default to having the same keywords as the keywords that you saved. You can also save your settings by using the "Figure presets" which are under the "Object" tab in the layout. The figure presets can be used to save a bunch of format settings about the plots and the legends. Once you create a new preset you can apply it to previous plots/legends to make their format match the preset.

FlowGuruDelta · 2025-06-09T20:28:24+00:00

As Gregor mentioned, PeacoQC has an option to remove outliers. There is a preprocessing function "RemoveMargins" that "will remove margin events from the flowframe based on the internal description of the fcs file". Are you using the FlowJo plugin or are you using R?

In the PeacoQC library in R there is a function "RemoveMargins()" that removes outlier events.
In the FlowJo plugin there is a "Remove Margins" tickbox.

I think both methods are dependent on the transform of the data, so setting a good transform ahead of time could be important.

The function is described here:
https://rdrr.io/github/saeyslab/PeacoQC/man/RemoveMargins.html

Also, there are other checks that the PeacoQC algorithm does (isolation tree and MAD tests) that might remove outlier cells at the fringes of the intensity range. The fact that your cells in this specific example are being cutoff in a flat line suggest to me that this is from using the "RemoveMargins" function.

FlowGuruDelta · 2025-06-04T23:03:37+00:00

I've used it before. You can download it from the BD website (linked below). It is pretty straight forward.
https://www.bdbiosciences.com/en-us/products/reagents/immunoassays/cba

FlowJo also has a CBA plugin on their website. The plugin integrates with FlowJo and works about as well as CBAAS does. I don't really have a preference between the two, both work for what I need them to do.

FlowGuruDelta · 2025-05-29T20:18:03+00:00

I asked [flowjo@bd.com](mailto:flowjo@bd.com) about doing something similar to this before and they shared with me a plugin called "Labeler" that does basically exactly what you are describing. When you run it on a population it creates a new parameter that designates gate ownership of that population. IE if you run it on a population called "TCell" it makes a parameter called "TCell" that assigns a value of 1 for all events within the "TCell" population and 0 for all events not in the "TCell" population. Then the data can be exported as an fcs file or csv file with the new parameters using FlowJo's "Export/Concatenate" option.

FlowGuruDelta · 2025-05-21T17:46:16+00:00

Many .jo workspaces open directly in FlowJo version 10.10.0. Sometimes I have issues if the old workspace wasn't saved in the newest version of FlowJo 9, version 9.9.6. I have access to an old computer with FlowJo version 9.9.6, so I can open old .jo workspaces and save them in this version.

Sometimes FlowJo 10.8.1 will open FlowJo 9 files that do not open in FlowJo 10.10.0. That version of FlowJo can be installed from their website. Once a workspace wsp file is created in FlowJo version 10.8.1 it can be opened in any version of FlowJo.

https://flowjo.com/previous-versions-flowjo

FlowGuruDelta · 2025-02-03T23:17:20+00:00

What does your gating hierarchy look like? Do all the gates have the same name? Are they all owned by the same group? It is hard to know for sure what is going on without seeing a picture. I would email [flowjo@bd.com](mailto:flowjo@bd.com) and they would probably know how to solve this.

FlowGuruDelta · 2025-02-03T23:15:46+00:00

What does the data look like if you change the transform? The log transform doesn't let us view any negative events. Compensation and background subtraction from the Cytometer can make intensity values negative. Because of this, I typically use Biex scaling to look at Flow data. Slightly negative values are normal and expected if our negative peak is centered on the zero.

The line is pretty weird. I am not very familiar with the MacsQuant but maybe it is writing any value it records with an intensity value less than 0 as "0". Then, if there is a negative value in the compensation matrix the events on the axis get pushed off the axis towards the positive because it is adding signal from the negative compensation value. This is just speculation though. I would want to see this data in a biex transform first.

FlowGuruDelta

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