![]() ![]() The last step is to load the 96 FCS files into FlowJo v10.x.x and assign the Well ID keyword to each of the files corresponding to their position on the plate. Step 3: Using the plate layout to create a heatmap. So, in 10.0.8b, you can highlight all the populations and choose export (right click or within the File tab in the ribbon) and export this as 96 FCS files. So, if you can do this all in 10.0.8beta, that's your best bet (or on windows). You can do the export to 96 separate files in Mac version 9.8.3, but you can't do the initial script in 9.8.3. The problem here is that you can do the initial index sorting script in version 10.0.7, but you can't do the export to 96 FCS files in 10.0.7 for the Mac (I think you can do this in the windows version, but I'm not sure). Step 2: The next step is to export each of these populations as its own FCS file. If you end up with a bunch of "-" where it usually says the number of cells in each population, click the refresh button at the top of the workspace window and then it'll show you that there is 1 cell per region (Figure 2). You should now have 96 populations under your index sorting file. Highlight your index sorting file in the workspace and click the run button in the script editor window. You simply copy and paste the script starting with /** - Iterate samples - **/ all the way through gate.update() } and paste it into the script editor window (under the tools tab of the ribbon) in version 10.0.7 (if you have access to the 10.0.8beta version, I would do this step in that version.you'll see why later). Step 1: Use the Script Editor index sorting example from the Daily Dongle Blog (or see Addendum below regarding the method in Version 9). Running the initial script to create 96 populations Heatmap analysis of index sorting file.įigure 2. I'd really love for someone to tell me there is a way easier way to do this in FlowJo.įigure 1. What follows below is A method (not THE method) I stumbled upon to go from an index sort file from FACSDiVa to Figure 1 below. The goal of index sorting, in this case, is to retain the original FITC intensity information for each well after the sort. The resulting plate will be a mix of FITC low and FITC high clones. And here's the fun part.Īnalyzing index sorting data in FlowJo. I have an application where a user is index sorting based on a range of FITC intensities. There is a decent interface for looking at your index sort plate information within FACSDiVa, but if you're use to doing all your analysis is FlowJo, you probably want to bring that data over at some point. You can export both (or all) of these as FCS files. You should have a Pre-sort FCS file of your entire population, and an Index sort "tube" for each plate you ran. So, let's assume you figure out how to successfully perform an index sort in FACSDiVa. The second resource is a document presented at GLIIFCA 2014 by Matt Cochran (University of Rochester), in which he outlines some of his tips and tricks for working with index sorting in FACSDiVa 8. I only came upon this after our BD service engineer sent me a copy of it. The first, oddly enough, is BD's very own Index Sorting Manual (<- fixed bad link), which comes as an addendum to the FACSDiVa software manual and may not even be installed on your computer or available for download from BD's website. There are two resources that are quite helpful in figuring this part out. You may think index sorting is no more than checking a box in FACSDiVa, but there are enough one-off situations that arise that it really warrants a separate FAQ. Of course, I'm sure there are more elegant ways of doing this in other programs or even in FlowJo, but I needed this info yesterday, so I'm documenting it here for future reference.įiguring out index sorting in FACSDiVa. I've done the leg work already, so read on to get the info. They are out there, it's just a pain to jump around to various sites trying to compile all the information together. After scouring the web for resources on both index sorting and analyzing index sorting data outside of FACSDiVa, I decided to compile all the resources in one place. Getting used to a brand new set of DiVa issues and quirks has been difficult, but we soldiered on nonetheless. We recently upgraded our FACSAria to FACSDiVa 8 running on Windows 7 primarily for the ability to do index sorting. ![]()
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