FlowJo [CELL CYCLE]

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Күн бұрын

Are you new to data analysis in FlowJo or looking for a starting point? This video will give you a quick overview on analyzing your cell cycle data in FlowJo!
Looking for more FlowJo content? Let us know in the comments what other FlowJo content you would like covered!

Пікірлер: 52

@djhell3054 2 жыл бұрын

Thank you so much! You are the best. Please keep making these videos.

@pedrocoelho6450 Жыл бұрын

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?

@ajarieger_flow Жыл бұрын

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

@yoniaa873 10 ай бұрын

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?

@ajarieger_flow 10 ай бұрын

I wouldn’t recommend it- you’ll need to show where those boundaries were drawn in order to properly present your data

@manpreet_kaur_gill_94 4 ай бұрын

Hi, Can you tell me how you manually drew the range and skipped using a model?

@ohoudalharbi4424 2 жыл бұрын

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??

@ajarieger_flow 2 жыл бұрын

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.

@manpreet_kaur_gill_94 4 ай бұрын

Hi A.R., Is it possible to reuse the gating done during acquisition in flowjo?

@ajarieger_flow 4 ай бұрын

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

@job506 Жыл бұрын

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?

@ajarieger_flow Жыл бұрын

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.

@job506 10 ай бұрын

@@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.

@_clarisse_manishimwe 6 ай бұрын

Hello Dr Rieger, I have a question concerning Fluorescence Minus One (FMO) controls. Are they necessary when analyzing cell cycle in FlowJo?

@ajarieger_flow 4 ай бұрын

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.

@_clarisse_manishimwe 4 ай бұрын

@@ajarieger_flow thank you, Dr Rieger

@job506 4 ай бұрын

Hi A.R.: @0.41s, please how can I reduce the percentage of cell on the chart edges? Many thanks.

@job506 13 күн бұрын

....this should be resolved at the point of data acquisition.

@jimmievu Жыл бұрын

Hello, could you also make a tutorial on cell cycle analysis with EdU plus PI labeling?

@ajarieger_flow Жыл бұрын

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

@macadance92 Жыл бұрын

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

@ajarieger_flow Жыл бұрын

Hi! Sorry I missed this comment!! Can you give me a few more details about your issues?

@macadance92 Жыл бұрын

@@ajarieger_flow we sorted it out. It was a wrong voltage issue.

@ajarieger_flow Жыл бұрын

@@macadance92 great! I'm glad you got it sorted! Sorry again for missing your earlier comment.

@armandoes 2 жыл бұрын

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?

@ajarieger_flow 2 жыл бұрын

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.

@job506 13 күн бұрын

@@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... kzfaq.info/get/bejne/mNpjltSSzczIfas.html

@dailytrips265 2 жыл бұрын

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?

@ajarieger_flow 2 жыл бұрын

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 :)

@_clarisse_manishimwe 7 ай бұрын

@@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 :)

@ajarieger_flow 7 ай бұрын

@@_clarisse_manishimwe yes exactly! I'm glad you've found them useful :)

@job506 14 күн бұрын

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.

@ajarieger_flow 9 күн бұрын

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.

@job506 8 күн бұрын

@@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?

@ajarieger_flow 8 күн бұрын

@@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

@job506 8 күн бұрын

@@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.

@ajarieger_flow 8 күн бұрын

@@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

@user-ju3xu9kv3w 8 ай бұрын

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?

@user-ju3xu9kv3w 8 ай бұрын

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

@ajarieger_flow 8 ай бұрын

So I better know what’s going on- what feature is missing? Are you missing the W signal? Or something else?

@user-ju3xu9kv3w 8 ай бұрын

W signal is missing. I am using PE-A and PE-H for analysis. I am not getting my results?

@ajarieger_flow 8 ай бұрын

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

@user-ju3xu9kv3w 8 ай бұрын

@@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

@job506 10 ай бұрын

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?

@ajarieger_flow 10 ай бұрын

You can reach me at aja@ualberta.ca

@job506 10 ай бұрын

@@ajarieger_flow Thank you very much. You're simply so amazing!