a colleague of mine has created a template for FACS analysis (attached). Her aim was to have the possibility to analyze different gatings in one sample. However, if one loads a dataset in all three columns (see images attached) and changes one gating, this applies to all three columns. The idea was to have three different gatings in one row.
Is there a way to change the template so that it works the desired way?
I checked the FCX file. I think there is a misunderstanding between gates and regions.
FCSalyzer has a different concept than FlowJow or FACS DIVA; it follows the concept of the old CellQuest software. In short, there are regions to marks cellpopulations, and then gates that are built from the regions to filter the data for particular plots.
I see in the template five regions: R.singlets, R.sperm, R.DFI, R.spermRef, R.DFI-Ref. Those are used to define five gates: G0 (R.singlets), G1 (R.sperm AND R.singlets), G2 (R.DFI), G3 (R.spermRef), G4 (R.DFI-Ref).
So based on your description, I would have expected three columns (one column = two document pages), with a different gate(!) for each column. However, instead in the first row, all three columns have the same gate G3 (on the large plot with the statistics; the small plots have no gate applied). You can see that in the screenshots, as the statistics box in all three columns says "gate: G3". On the second row, only the first large plot has G3, all other large plots in that row have G1. In the subsequent rows, all plots have G1.
Your colleague needs to set different gates on the different columns.
Also, regions are independent of other regions and gates, but gates are dependent on regions (as they are defined by the regions). This impacts the effects of gating and also the statistics. Shown on the plots are regions. If a region changes (like between screenshot 1 and 2), all gates that contain the region in their definition will change as well. In the screenshot, "R.DFI-Ref" was changed and thus G4 as well (however, as G4 is not used in the template this change is not apparent). In addition, the statistics that show the events contained in a region(!) also change their values for R.DFI-Ref. This is correct and expected behaviour.
It is difficult for me to exactly understand what your colleague tried to achieve. But in principle she would need to define the three gates for filtering the events to her needs. Then, she would need to apply one gate for each columns. The statistics would then show the events that fall into a region, after the datset events were filtered by the gate.
[An example: maybe the columns are meant to analyse R.sperm, R.DFI, R.sperm-REF and R.DFI-Ref - but that would require four columns. Maybe all events should also be singlets, in other words be contained in the region R.singlets. If so, four gates would be defined: G0 (singlets AND R.sperm), G1 (singlets AND R.DFI), G2 (singlets AND R.sperm-REF), G3 (singlets AND R.DFI-Ref). Then, the four gates would be applied to a separate column each, and the statistics in the columns would then show the data for the five regions after the events were filtered by the respective gate. Please note that the statistics tables would say "Gate: G0", "Gate: G1", "Gate: G2", "Gate: G3", respectively.]
I hope this helps?
Regards,
Sven
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thank you very much for your fast and comprehensive reply! I have spoken to my colleague, and you pointed out what's wrong. She chose regions, but not different gates and can thus alter this. Thanks!
Best regards,
Sebastian
If you would like to refer to this comment somewhere else in this project, copy and paste the following link:
Hey community,
a colleague of mine has created a template for FACS analysis (attached). Her aim was to have the possibility to analyze different gatings in one sample. However, if one loads a dataset in all three columns (see images attached) and changes one gating, this applies to all three columns. The idea was to have three different gatings in one row.
Is there a way to change the template so that it works the desired way?
Best regards,
Sebastian
Last edit: Sebastian 2020-06-13
Hi Sebastian,
I checked the FCX file. I think there is a misunderstanding between gates and regions.
FCSalyzer has a different concept than FlowJow or FACS DIVA; it follows the concept of the old CellQuest software. In short, there are regions to marks cellpopulations, and then gates that are built from the regions to filter the data for particular plots.
I see in the template five regions: R.singlets, R.sperm, R.DFI, R.spermRef, R.DFI-Ref. Those are used to define five gates: G0 (R.singlets), G1 (R.sperm AND R.singlets), G2 (R.DFI), G3 (R.spermRef), G4 (R.DFI-Ref).
So based on your description, I would have expected three columns (one column = two document pages), with a different gate(!) for each column. However, instead in the first row, all three columns have the same gate G3 (on the large plot with the statistics; the small plots have no gate applied). You can see that in the screenshots, as the statistics box in all three columns says "gate: G3". On the second row, only the first large plot has G3, all other large plots in that row have G1. In the subsequent rows, all plots have G1.
Your colleague needs to set different gates on the different columns.
Also, regions are independent of other regions and gates, but gates are dependent on regions (as they are defined by the regions). This impacts the effects of gating and also the statistics. Shown on the plots are regions. If a region changes (like between screenshot 1 and 2), all gates that contain the region in their definition will change as well. In the screenshot, "R.DFI-Ref" was changed and thus G4 as well (however, as G4 is not used in the template this change is not apparent). In addition, the statistics that show the events contained in a region(!) also change their values for R.DFI-Ref. This is correct and expected behaviour.
It is difficult for me to exactly understand what your colleague tried to achieve. But in principle she would need to define the three gates for filtering the events to her needs. Then, she would need to apply one gate for each columns. The statistics would then show the events that fall into a region, after the datset events were filtered by the gate.
[An example: maybe the columns are meant to analyse R.sperm, R.DFI, R.sperm-REF and R.DFI-Ref - but that would require four columns. Maybe all events should also be singlets, in other words be contained in the region R.singlets. If so, four gates would be defined: G0 (singlets AND R.sperm), G1 (singlets AND R.DFI), G2 (singlets AND R.sperm-REF), G3 (singlets AND R.DFI-Ref). Then, the four gates would be applied to a separate column each, and the statistics in the columns would then show the data for the five regions after the events were filtered by the respective gate. Please note that the statistics tables would say "Gate: G0", "Gate: G1", "Gate: G2", "Gate: G3", respectively.]
I hope this helps?
Regards,
Sven
Dear Sven,
thank you very much for your fast and comprehensive reply! I have spoken to my colleague, and you pointed out what's wrong. She chose regions, but not different gates and can thus alter this. Thanks!
Best regards,
Sebastian