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Event Length settings in CyTOF/Flowjo softwares

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SaraSuliman

Participant

Posts: 2

Joined: Fri Jan 31, 2014 12:19 pm

Post Mon May 12, 2014 3:17 pm

Event Length settings in CyTOF/Flowjo softwares

Hi,
We recently acquired CyTOF2 in Cape Town. After performing a standard experiment of PBMC stimulation with mitogen, the Event_length parameter on flowjo was squished to the axis. We got 2 blank templates from CyTOF1 users at SIgN which gave us a range of 0-40K (in increments of 10K) and 0-4K (in increments of 1000). However, in both cases the events were squished to the left on the Event_Length parameter on flowjo. We want to know whether this is a problem of the settings on the CyTOF software, or something that can be resolved with the settings on flowjo, or if it was a consequence of the acquisition speed on the instrument. The pushes looked distinct on the rain display during acquistion, so it's difficult to assume that was the issue, we estimated approximately between 300 and 500events/sec on the different samples, all of which showed the same squished display. Any advice would be appreciated.

Thanks!
Sara
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Tue May 13, 2014 2:24 pm

Re: Event Length settings in CyTOF/Flowjo softwares

Hi Sara,
You will have to change your display settings in FlowJo. Event lengths for cells fall between 10 and 150, with most being around 20-30. You will want to display a range of 0-300 or something similar. Generally this is done on a linear axis.
If you let us know what version of FlowJo you are using we can guide you on how to change the display settings. Usually clicking the "T" button on the plot axis will allow you to do this, otherwise its in preferences.
Rob
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue May 13, 2014 3:32 pm

Re: Event Length settings in CyTOF/Flowjo softwares

Hi Sara,

Mac FlowJo X allows you to switch scaling type (linear vs log, etc) and length of axes (1e4, 1e5, etc). You'll want to have Time and Event_length (or Cell_length) in Linear scaling.


However, if you're using a Mac FlowJo 9.x, you'll have to change your Preferences. I have attached a screenshot of what I have my settings at (currently using 9.7.5).

1. Open FlowJo. Close the empty Workspace that appears.
2. FlowJo>Preferences. In the lower right, check the CyTOF box, and have log parameter range set to 20000 (perhaps 9000, if you're using a CyTOFv2).
3. Click "Define" to open the right-hand pane in the attached picture. Add Time and Cell_length to the linear box.
4. I think it's the Width basis that helps shove the plots off the origin (up, and to the right) so you can more easily draw gates on the negative populations.
5. Hit OK on the 2nd window to close it.
6. At the middle bottom, click the dropdown menu. Select "Save This Set of Preferences", name it, and save.
7. FlowJo>Switch To Preference Set. Select your new set of CyTOF preferences.
8. Now open a new Workspace.

In all the v9.x versions of FlowJo I've used, I've *never* been able to get it to successfully switch Preference types while a Workspace window is open. So, you really do need to close that initial workspace, switch your preferences, then open a new one. Once you have successfully created a CyTOF-related workspace, then you can reopen that workspace for new files.


Mike
Attachments
CyTOF FlowJO settings-102711.png
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Evan

Participant

Posts: 5

Joined: Sat Nov 30, 2013 4:56 am

Post Wed May 14, 2014 12:54 am

Re: Event Length settings in CyTOF/Flowjo softwares

Hi Sara,

I wonder if the problem is because the "Cell_length" parameter name for the cytof2 software has been changed to "Event_length" and this is why FlowJo 9 with your settings is not forcing it into a linear scale (see upper right side of the screen-shot that Mike posted)? Perhaps you can try changing to this. I uploaded a screen shot with settings where I tried changing this as suggested. Maybe it will work if this is correct...

These settings (other than the change to "Event_length" from "Cell_length") are what we have been using in flowjo9.7.5 for cytof1 data using relatively recent cytof1 acquisition software (with updated and corrected dual count calibration, and a lower top of scale that we used with previous versions of the software).

I am still using FlowJo9.7.5 instead of FlowJo10 because I prefer the logicle (biexponential) transform over the log or arcsinh transforms. I suppose this is a matter of preference, but there may also be some mathematical or statistical reasons for this as well (see Parks et al Cytometry A 2006, Moore et al., Cytomtery A 2012, etc). Perhaps this is debatable. Does anybody know if it is possible to use the logicle transform using FlowJo10? - if so I can not figure out how to do this.

Thanks,
Evan
Attachments
Flowjo settings to try.png
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nlazarus

Contributor

Posts: 20

Joined: Wed Nov 13, 2013 7:28 pm

Post Wed May 14, 2014 1:17 am

Re: Event Length settings in CyTOF/Flowjo softwares

I was having the same exact problem and learned the following trick from those at the Stanford FACS facility. (flowjo version 9.7.5)

Open a workspace with a CyTOF file in the workspace. Click on the CyTOF fcs file within the workspace.

Under "Platform" pull down menu, select "Derive Parameters" then "Define New or Change"

The cursor will automatically be in the "Custom Derived Parameters" box. Just below that, there is a pull down menu "insert parameter".
Select "Event_Length".
Click the box below "Display with linear scale".
Just above, "display axis: Highest Value". Set it to a value (I use 150).
Below, there is a field "New parameter name". Input anything you like to name the parameter (I use Event_ADJ).
Click the box "Add algebraic parameter". Click "Done"

Back in the workspace, click on one axis of your fcs files as though you were going to change it....at the very bottom of the list of parameters, the Event_ADJ should be an option. Select that. Your problem should be solved.

See attachment.
Attachments
Screen shot 2014-05-13 at 6.15.09 PM.png
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SaraSuliman

Participant

Posts: 2

Joined: Fri Jan 31, 2014 12:19 pm

Post Wed May 14, 2014 1:05 pm

Re: Event Length settings in CyTOF/Flowjo softwares

Phenomenal suggestions!

Evan, we are using flowjo 9 as well. I agree that flowjo 10 doesn't display data nicely. If we use your template and apply a new derived parameter from platform as Mike and Nicole suggested, it works very well to spread the event length but then it shows the events as spikes at each increment of 10. When I start with flowjo default settings and just of course click CyTOF in the settings, and add a new derived parameter to look at event length (max of 150 as suggested), it works very well to spread out the events in that parameter.

Still early days in the analysis and troubleshooting- so I will likely be posting more questions soon!
Thanks for the suggestions everyone!
Sara

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