Hi Aja: Many thanks for the video. Please what determines the gating coverage? At 0:38 sec, what happens to the other cells far up? Why were they excluded?
The cells along the far edge of the FSC/ SSC plot are very likely clumps/doublets which is why I have excluded them. You could also gate them in the remove them in the doublets gate.
@@ajarieger_flow Many thanks, ma. I have another observation too: the summation of your cell cycle statistics is more than 100%. I have experienced this in my analyses too and even had negative % values. Please, how do I explain these? Best regards.
Thank you for this short and succinct video! I still have a question regarding cell cycle analysis. When running my samples, I always try to align G0/G1 peak to 200k on x-axis (linear scale). Still, most of the time G0/G1 peaks from multiple samples do not align to this region on the axes which is problematic when I try to overlay these histograms together. How would you go about fixing this? Would you adjust the voltage for each sample to align G0/G1 peak to 200k on x-axis before recording or can this be somehow fixed in FlowJo? Some of my samples are really off, especially the ones containing less cells. Thank you!
Great question!! This usually is a protocol issue (do NOT adjust your voltage for each sample). Instead, I would go about making sure (1) you have titrated your stain to ensure you are at a saturating concentration in all your samples and (2) ensure you are staining for long enough. (Both these will also help improve CV's). Cell cycle, on paper, seems like a very simple protocol that should be easily mastered but there are a lot of intricacies that make it one of the most challenging protocols out there
Thanks for reaching out! I unfortunately don't have any FCS files for this type of experiment. If you're willing to send some my way, I would be happy to put this together. aja@ualberta.ca
Hey!! Thank you for this video. I have a question, couldn’t I exclude the doublets by gating FSC-H vs. FSC-A and then apply the cell cycle analysis to that population of single cells?
That’s a great question! With cell cycle, knowing accurately that your G2/M phase is only cells in that phase and not doublets is very important. So using the PI (or DNA dye you’re using) for doublets is better at ensuring this is the case
Hi Aja, many thanks again for this video. Please, under what circumstance is it necessary to constrain both G1 and G2 peaks of the control sample for subsequently application of the settings to other test samples? In addition, is it ideal to use different models for the same set of samples as it was initially initiated for the sample that was not auto-analyzed? Best regards.
I would recommend you take a look at this: docs.flowjo.com/flowjo/experiment-based-platforms/cell-cycle-univariate/ You will need to use the same model to analyze all your samples.
@@ajarieger_flow Thank you so much again and again. It's getting more interesting considering the fact that the values %G1 obtained for the controls are 33.4, 15.1, and 24.9, respectively. One would naturally have imagined that the values seem significantly different; how should this be explained, please?
@@job506 have you synchronized your cells? You can get a lot of variability without synchronization. If you’re not synchronizing, you will need more replicates
@@ajarieger_flow I was actually referring to the data presented in the link you sent. In my experiments, I did not synchronize the cells. Please what approach is better for synchronizing cells? Thank you so much.
@@job506 there are a lot of methods. This gives a good overview: en.m.wikipedia.org/wiki/Cell_synchronization I’ve done a double thymidine block in the past
Thank you for this video. If you've manually drawn and analyzed peaks in the G1 and G2 regions that cannot be automated, is it possible to remove only the labeling lines for G1 and G2, similar to other figures in research papers?
Sorry I missed this comment! Generally not as the staining pattern is very clear. If you are adding additional markers to analyze specific parts of the cell cycle, you may.
Thank you so much for the video. It is really useful. I have analysed my results but I have a problem that the cell cycle’s histograms do not have the same cell number on the Y axis?? Do you have any suggestions??
Thanks for reaching out! Generally there is no issue with there being different numbers on the Y axis. However, you can play around with normalizing histograms if you are doing overlays etc.
You can- if you’re using diva for acquisition. This will show you how: docs.flowjo.com/flowjo/workspaces-and-samples/diva-integration/ If you’re using another acquisition software, it’s not possible
Hi, quick question: I'm interested in the analysis of SubG0. Why not incorporate that in the cell cycle model? There is a "less than G0" tag. Also, shouldnt you apply the "G1" region to all the histograms and not just the one that cant be automatically modelled?
Yes you can use the less than G0 tag- I have just not had good luck with it. The G1 region is not a region in the same way as a gate is (which you should apply across all samples); it is more a way to show the algorithm where the G1area is. Since this will be potentially in a slightly different spot in your other samples, the same constraints may not work.
@@ajarieger_flow Thank you, Dr. Rieger. However, the Application Scientist at FlowJo indicates that the constraints should be applied using the settings with the control sample... ruclips.net/video/bt3cqahfdMw/видео.html
Thanks for reaching out! If PI-W is not on your list, that means that it was not selected when you collected your samples. There is unfortunately no way to change this now. In the future, if you select PI-W in your parameter list, you'll be all set :)
@@ajarieger_flow Hello Dr Rieger. Do you mean that when running samples using a flow cytometer, we should check the parameters list and select PI-W? Thank you for the excellent tutorials :)
This parameter needs to be added during acquisition. There’s nothing you can do in analysis to add it. However, you can still do double discrimination with PE-A vs PE-H (similar to how it’s done with FSC) and in some instruments this actually provides better discrimination
@@ajarieger_flow thank you i will again do it follow your videos. Can i be in touch if i face some problem through your email? If possible please your email
Hi A.R., Multiple thanks to you again. Please, could you kindly assist me with your email address for further clarifications with my annexin V-FITC plots?
![FlowJo [tSNE]](http://i.ytimg.com/vi/UK725D14-bo/mqdefault.jpg)
![FlowJo [tSNE]](/img/tr.png)
![FlowJo [APOPTOSIS ANALYSIS]](http://i.ytimg.com/vi/cp0QlV5cTFk/mqdefault.jpg)
Thank you so much! You are the best. Please keep making these videos.
Hi Aja: Many thanks for the video. Please what determines the gating coverage? At 0:38 sec, what happens to the other cells far up? Why were they excluded?
The cells along the far edge of the FSC/ SSC plot are very likely clumps/doublets which is why I have excluded them. You could also gate them in the remove them in the doublets gate.
@@ajarieger_flow Many thanks, ma. I have another observation too: the summation of your cell cycle statistics is more than 100%. I have experienced this in my analyses too and even had negative % values. Please, how do I explain these? Best regards.
Thank you for this short and succinct video! I still have a question regarding cell cycle analysis. When running my samples, I always try to align G0/G1 peak to 200k on x-axis (linear scale). Still, most of the time G0/G1 peaks from multiple samples do not align to this region on the axes which is problematic when I try to overlay these histograms together. How would you go about fixing this? Would you adjust the voltage for each sample to align G0/G1 peak to 200k on x-axis before recording or can this be somehow fixed in FlowJo? Some of my samples are really off, especially the ones containing less cells. Thank you!
Great question!! This usually is a protocol issue (do NOT adjust your voltage for each sample). Instead, I would go about making sure (1) you have titrated your stain to ensure you are at a saturating concentration in all your samples and (2) ensure you are staining for long enough. (Both these will also help improve CV's). Cell cycle, on paper, seems like a very simple protocol that should be easily mastered but there are a lot of intricacies that make it one of the most challenging protocols out there
Hello, could you also make a tutorial on cell cycle analysis with EdU plus PI labeling?
Thanks for reaching out! I unfortunately don't have any FCS files for this type of experiment. If you're willing to send some my way, I would be happy to put this together. aja@ualberta.ca
Thanks so much for this video! I was wondering if there are any tricks or musts during data acquisition. My samples just create one single peak
Hi! Sorry I missed this comment!! Can you give me a few more details about your issues?
@@ajarieger_flow we sorted it out. It was a wrong voltage issue.
@@macadance92 great! I'm glad you got it sorted! Sorry again for missing your earlier comment.
Hey!! Thank you for this video. I have a question, couldn’t I exclude the doublets by gating FSC-H vs. FSC-A and then apply the cell cycle analysis to that population of single cells?
That’s a great question! With cell cycle, knowing accurately that your G2/M phase is only cells in that phase and not doublets is very important. So using the PI (or DNA dye you’re using) for doublets is better at ensuring this is the case
Hi A.R.: @0.41s, please how can I reduce the percentage of cell on the chart edges? Many thanks.
....this should be resolved at the point of data acquisition.
Hi Aja, many thanks again for this video. Please, under what circumstance is it necessary to constrain both G1 and G2 peaks of the control sample for subsequently application of the settings to other test samples? In addition, is it ideal to use different models for the same set of samples as it was initially initiated for the sample that was not auto-analyzed? Best regards.
I would recommend you take a look at this:
docs.flowjo.com/flowjo/experiment-based-platforms/cell-cycle-univariate/
You will need to use the same model to analyze all your samples.
@@ajarieger_flow Thank you so much again and again. It's getting more interesting considering the fact that the values %G1 obtained for the controls are 33.4, 15.1, and 24.9, respectively. One would naturally have imagined that the values seem significantly different; how should this be explained, please?
@@job506 have you synchronized your cells? You can get a lot of variability without synchronization. If you’re not synchronizing, you will need more replicates
@@ajarieger_flow I was actually referring to the data presented in the link you sent. In my experiments, I did not synchronize the cells. Please what approach is better for synchronizing cells? Thank you so much.
@@job506 there are a lot of methods. This gives a good overview: en.m.wikipedia.org/wiki/Cell_synchronization
I’ve done a double thymidine block in the past
Thank you for this video.
If you've manually drawn and analyzed peaks in the G1 and G2 regions that cannot be automated, is it possible to remove only the labeling lines for G1 and G2, similar to other figures in research papers?
I wouldn’t recommend it- you’ll need to show where those boundaries were drawn in order to properly present your data
Hi,
Can you tell me how you manually drew the range and skipped using a model?
Hello Dr Rieger, I have a question concerning Fluorescence Minus One (FMO) controls. Are they necessary when analyzing cell cycle in FlowJo?
Sorry I missed this comment! Generally not as the staining pattern is very clear. If you are adding additional markers to analyze specific parts of the cell cycle, you may.
@@ajarieger_flow thank you, Dr Rieger
Thank you so much for the video. It is really useful. I have analysed my results but I have a problem that the cell cycle’s histograms do not have the same cell number on the Y axis??
Do you have any suggestions??
Thanks for reaching out! Generally there is no issue with there being different numbers on the Y axis. However, you can play around with normalizing histograms if you are doing overlays etc.
Hi A.R.,
Is it possible to reuse the gating done during acquisition in flowjo?
You can- if you’re using diva for acquisition. This will show you how: docs.flowjo.com/flowjo/workspaces-and-samples/diva-integration/
If you’re using another acquisition software, it’s not possible
Hi, quick question: I'm interested in the analysis of SubG0. Why not incorporate that in the cell cycle model? There is a "less than G0" tag. Also, shouldnt you apply the "G1" region to all the histograms and not just the one that cant be automatically modelled?
Yes you can use the less than G0 tag- I have just not had good luck with it. The G1 region is not a region in the same way as a gate is (which you should apply across all samples); it is more a way to show the algorithm where the G1area is. Since this will be potentially in a slightly different spot in your other samples, the same constraints may not work.
@@ajarieger_flow Thank you, Dr. Rieger. However, the Application Scientist at FlowJo indicates that the constraints should be applied using the settings with the control sample... ruclips.net/video/bt3cqahfdMw/видео.html
Thank you so much, I have a problem that is I don't find the PI-width parameter whereas I have only FSC-width so can I use it?
Thanks for reaching out! If PI-W is not on your list, that means that it was not selected when you collected your samples. There is unfortunately no way to change this now. In the future, if you select PI-W in your parameter list, you'll be all set :)
@@ajarieger_flow Hello Dr Rieger. Do you mean that when running samples using a flow cytometer, we should check the parameters list and select PI-W? Thank you for the excellent tutorials :)
@@_clarisse_manishimwe yes exactly! I'm glad you've found them useful :)
How can i download the functions in my Flow 10.9.0 as in the video the function you used not showing in my software?
I am using FSC against SSC and then PE-H with PE-A but not getting fruitful results. Your method function not showing in my flow jo
So I better know what’s going on- what feature is missing? Are you missing the W signal? Or something else?
W signal is missing. I am using PE-A and PE-H for analysis. I am not getting my results?
This parameter needs to be added during acquisition. There’s nothing you can do in analysis to add it. However, you can still do double discrimination with PE-A vs PE-H (similar to how it’s done with FSC) and in some instruments this actually provides better discrimination
@@ajarieger_flow thank you i will again do it follow your videos. Can i be in touch if i face some problem through your email? If possible please your email
Hi A.R., Multiple thanks to you again. Please, could you kindly assist me with your email address for further clarifications with my annexin V-FITC plots?
You can reach me at aja@ualberta.ca
@@ajarieger_flow Thank you very much. You're simply so amazing!