Thank you very much for the great tutorial! I'm working with FACS data similar to yours and I have 2 groups which I want to compare. I gave them key words and then concatenated all of them together. Now I want to run UMAP. Do I have to run UMAP on the concat file (as you did) or do I have to gate on the different groups first and then run UMAP on every single group individually?
Thank you very very much for your rapid answer!!!! That helps a lot already. For the concatenation step I am unsure wheter to use concat populations or concat group?
If I concatenate the FCS files of the 5 contols with the 5 treated samples in Flowjo there is a option to choose between "Export/Concatenate Populations" or "Export/Concatenate Group". Which one would you recommend to use. After that I want to run the UMAP. And then I want to have a look at the lymphocytes of the control vs. The treated group.
Thank you for the wonderful tutorial, may I ask why are we excluding CD45 for UMAP generation? I understand L/D but don't know why we are not taking CD45.
Thanks for asking! I didn't include it as it wasn't important to the biology I was looking at. This is something you should test with your samples- if including or excluding gives you better clustering.
Hi Aja, thank you for sharing this. Is there a way to do it on flowjo, so that when we concatenated a group of patients based on their conditions, and we can see highlight on CD4 (example) and showing data per patient on the same UMAP? Thank you!
There is! Before concatenation, give each group a keyword (usually 1, 2, 3, etc). Afterwards you can plot that keyword (use a linear scale) and gate them. Keyword 1 (group 1) will be at 1000, etc. You can then use these gates as you would any other gate. Hope that helps!
@@ajarieger_flow Thank you so much for your reply. I did assign a keyword using numerical numbers, and gate them using a linear scale. But I am unsure how to show that on the UMAP or diagram, may I ask if you would mind sharing more information about this? Thank you!
Hello, I working with cellls derived from different patients and we are characterizing all by some membrane marker expression. We wanted to create a UMAP based on the exprresion of different markers, i would like to know if we can do that, thank you!
Hi! Thanks for reaching out. The expression of your markers is what the UMAP is using- you can select the markers to include or exclude when it set it up. Not sure if that answers your question- let me know if it doesn’t!
![FlowJo [CytoNorm]](http://i.ytimg.com/vi/1fJXxuEb-no/mqdefault.jpg)
![FlowJo [CytoNorm]](/img/tr.png)
![FlowJo [tSNE]](http://i.ytimg.com/vi/UK725D14-bo/mqdefault.jpg)
![Adjusting compensation: how to handle a "bad" matrix [FlowJo advanced]](/img/1.gif)
Thank you very much for the great tutorial!
I'm working with FACS data similar to yours and I have 2 groups which I want to compare. I gave them key words and then concatenated all of them together. Now I want to run UMAP. Do I have to run UMAP on the concat file (as you did) or do I have to gate on the different groups first and then run UMAP on every single group individually?
@@leniw.3659 I would run it on the full file so you can get all possible populations captured. You can then gate sub populations after
Thank you very very much for your rapid answer!!!! That helps a lot already. For the concatenation step I am unsure wheter to use concat populations or concat group?
@@leniw.3659 what do you mean by population vs group? I want to make sure we’re talking about the same thing
If I concatenate the FCS files of the 5 contols with the 5 treated samples in Flowjo there is a option to choose between "Export/Concatenate Populations" or "Export/Concatenate Group". Which one would you recommend to use.
After that I want to run the UMAP. And then I want to have a look at the lymphocytes of the control vs. The treated group.
I have a similar question and don't know whether I should choose the Export/concatenate group or Export/concatenate population option
Thank you for the wonderful tutorial, may I ask why are we excluding CD45 for UMAP generation? I understand L/D but don't know why we are not taking CD45.
Thanks for asking! I didn't include it as it wasn't important to the biology I was looking at. This is something you should test with your samples- if including or excluding gives you better clustering.
Hi Aja, thank you for sharing this. Is there a way to do it on flowjo, so that when we concatenated a group of patients based on their conditions, and we can see highlight on CD4 (example) and showing data per patient on the same UMAP? Thank you!
There is! Before concatenation, give each group a keyword (usually 1, 2, 3, etc). Afterwards you can plot that keyword (use a linear scale) and gate them. Keyword 1 (group 1) will be at 1000, etc. You can then use these gates as you would any other gate. Hope that helps!
@@ajarieger_flow Thank you so much for your reply. I did assign a keyword using numerical numbers, and gate them using a linear scale. But I am unsure how to show that on the UMAP or diagram, may I ask if you would mind sharing more information about this? Thank you!
How to run Cytonorm, please.
Stay tuned! I’ll try get that out this week
CytoNorm video out today!
Hello, I working with cellls derived from different patients and we are characterizing all by some membrane marker expression. We wanted to create a UMAP based on the exprresion of different markers, i would like to know if we can do that, thank you!
Hi! Thanks for reaching out. The expression of your markers is what the UMAP is using- you can select the markers to include or exclude when it set it up. Not sure if that answers your question- let me know if it doesn’t!