I want to compare how the cell population is changing from time zero through the process, for which I want to see the chnage in the FSC histogram for each sample with respect to the time zero. I manage to get the overlay histograms individually, but it would be nice to have them all together. Using Image collector, I can only collect the individual histograms, not the overlays. Any help?
Thanks! :)
Hi Sumana! I'm using Flowing software too and I found a solution to your problem :)
- Open your FCS files for time zero and first time point in separate Dot Plots. let's call Dot Plot at time zero DP0 and Dot plot at first time point DP1.
- Create a Histogram for each Dot Plot. Let's call them H0 (time zero) and H1 (first time point).
- Open Overlay Histogram menu (right click on Overlay Histogram), go to "Define the master histogram" and select H1.
- Connect Overlay Histogram with Image Collector. Image Collector will be empty at this point.
- Click on DP0. The first overlay should appear in Image Collector.
- Click on DP1 and open FCS file for the next time point. Overlay will show the right histograms, however, you won't see it in Image Collector.
- Click on DP0 again. Now you should see the second overlay next to the first one.
- Repeat the last two steps until you have all the overlays in Image Collector.
I hope this trick will work for you as it has worked for me. Let me know if it won't and I will send you my analysis file.
Best,
Klemen
Hi Klemen Dolinar
Thanks a lot for the helpful input!! It works - except that I had to define the H1 histogram as the Master histogram for the overlay. Neat trick :)
Sudeep Kumar Thanks, but unfortunately FlowJo is not free!
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