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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

@@kaybash21 you can treat it the same way, you just won’t have an unstained on your plot. You can still do stain index etc as normal

Thanks! I appreciate it! I'm always collecting topics- if there is something specific, let me know! Might put some more together soon :)

I've not gotten that warning before... I would follow the directions and contact FlowJo help if it still keeps happening.

@@ajarieger_flow Thank you so much! I am having another problem when running the FlowSOM. After calculating for log time it says "Failed to creat output"

@@ireneraposogutierrez779 I have had this one- a restart fixed it. You can also try removing and reinstalling the plugin

@@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

@@ZzZ-kj9qf nothing really- just a personal preference! As long as you choose one and use it consistently, you’re all set!

@@ajarieger_flow Ok got it, thank you for the quick reply!

@@prasanthchakrapani8105 this video will help: th-cam.com/video/e6k5fD1w2RU/w-d-xo.htmlsi=GCjzcXmaQIlIclJP

@@smritimishra4609 thanks a great question! I don’t know the answer but I assume it’s largely due to operator impatience

Hi! First question- have you titrated all your antibodies? Normally shifting population backgrounds are due to too much antibody being present.

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

That's what the stain index is for! Whichever has the highest stain index is your sample with the best resolution.

Great to hear from you! I kept my titration video as simple as possible and didn’t have a viability stain in it so there wasn’t the added complication of compensation. However, best practice is to include it, as you have done, so you can eliminate dead cells and thereby choose the correct concentration. On the Aurora, you have a couple of options: 1. You can make single colour controls and unmix before doing your analysis. In this case, you’d need a different experiment for each colour you’re titrating so each has their own matched unmixing matrix. Or 2. So long as you stains are different enough (very different peak channels and distinct aspects) you can do your analysis on the raw data and avoid single colour controls and unmixing. Good luck and happy titrating!!

Glad this went through! It’s the first time I’ve seen it. Having acquired your experiment on the ImageStream, I would really highly recommend analyzing it in IDEAS (the ImageStream software) rather than as an exported FCS in FlowJo. You get such a wealth of cell death information from the images, it would be a shame to lose out on that! IDEAS has a great analysis wizard that you can use to help out and reduce the learning curve for the software (it can be quite steep). If you do want to use FlowJo, the most similar axis labels would be: FSC = Area M01 (assuming this is your bright field channel) SSC = Intensity Ch 6 or 12 (depending on your configuration and how the data was acquired) AnxV = Intensity Ch 2 (assuming from your comment that you are using FITC) PI = Intensity Ch 4 All that being said- do the analysis in IDEAS! You will get so much more information out of it!

@@ajarieger_flow Thank you so much for the reply! You are such a lifesaver. I am aware of IDEAS, but the technical officer that provided me with some training with ImageStream told me IDEAS is a difficult software to use so I ended up looking for alternatives. Of which I stumbled across your channel and FlowJo seemed simple and intuitive enough so I went with FlowJo. Given your advise I'll definitely go back to IDEAS and see what I can achieve there. Hilariously enough even though my uni has a pretty advanced flow cytometer (ImageStream), very little people, even the technical officer herself, knows how to really operate the instrument to its full potential. The only unfortunate thing is that I acquired my data only in .fcs format, and not in .rif that IDEAS recognize. I tried importing my .fcs into IDEAS earlier and nothing really happened. I'll have to run my experiments again. But that's alright since its part of the learning process. In the meantime I'll mess around with FlowJo first with the help that you've provided. Again thank you so much for the help. Really really really appreciate your time. I hope you have a nice weekend!

​@@ajarieger_flow Oh my god I think YT deleted my comment AGAIN. I didn't include any links this time. YT please D: Anyway, thank you so so much for your help! You are such a lifesaver. I am aware of IDEAS. However, the technical officer who gave me some training to operate ImageStream told me the software is difficult to use (She doesn't even know how to use it herself). So I opted for alternatives. Of which I stumbled across your channel, and FlowJo seemed simple and intuitive enough so I decided on FlowJo. After reading your advise I'll definitely give IDEAS another go. Hilariously enough, even though my uni has a pretty advanced flow cytometer (The ImageStream), very few people, including the technical officer herself, know how to operate the instrument to its full potential. The only unfortunate thing is that I only acquired my data in .fcs format, and not in the .rif format that IDEAS mainly recognize. I tried to import my .fcs data into IDEAS but nothing really happened (Sometimes the software even crashes lmao). I'll have to run my experiments again but its not a big deal because I just did a trial run to get familiar with the process. In the meantime I'll mess around with FlowJo first. Again thank you so much for your help! I really appreciate your time and effort. Hope you have a nice weekend!

​ @ajarieger_flow Oh my god I think YT deleted my comment AGAIN. I didn't include any links this time. YT please D: Anyway, thank you so so much for your help! You are such a lifesaver. I am aware of IDEAS. However, the technical officer who gave me some training to operate ImageStream told me the software is difficult to use (She doesn't even know how to use it herself). So I opted for alternatives. Of which I stumbled across your channel, and FlowJo seemed simple and intuitive enough so I decided on FlowJo. After reading your advise I'll definitely give IDEAS another go. Hilariously enough, even though my uni has a pretty advanced flow cytometer (The ImageStream), very few people, including the technical officer herself, know how to operate the instrument to its full potential. The only unfortunate thing is that I only acquired my data in .fcs format, and not in the .rif format that IDEAS mainly recognize. I tried to import my .fcs data into IDEAS but nothing really happened (Sometimes the software even crashes lmao). I'll have to run my experiments again but its not a big deal because I just did a trial run to get familiar with the process. In the meantime I'll mess around with FlowJo first. Again thank you so much for your help! I really appreciate your time and effort. Hope you have a nice weekend!

Thanks!! I have a video on statistics- check that one out. If you still have questions, let me know

Weird- I've not seen that happen before. If cell source is working you should be fine. Unsure why FlowSOM keeps failing. I've found sometimes reinstalling the plugin can help. And make sure you are using the correct version of R

Thanks for reaching out! Sorry about the delay in getting back- I missed seeing this comment. Generally subtraction isn't recommended. Sometimes you will see people normalize the data to the control and look at fold change over the background. Your control also would benefit from having more than 51 total cells. This will give you a more robust population to work from in your comparisons.

It is a little bit of a chicken and the egg situation and really depends on the individual. Theoretically, if you are starting with titration and then voltration, all your samples are single stained so no compensation is needed. However, some users choose to test the full panel prior to this to ensure the staining and biology make sense- so in this case you may compensate prior to doing the voltration. When and how you choose to compensate is fully up to you and both are acceptable- I routinely compensate in FlowJo after acquisition (even if I have compensated during acquisition). As you say- it is just math. As long as your controls are appropriate and in place it doesn't matter when you compensate so long as you do it before drawing conclusions from the data.

@@ajarieger_flow Got it. Thank you so much for those insights! You're awesome!

Thanks for reaching out- I'm glad it was helpful for you. (1) My recommendation is always to start with all necessary controls and then determine what is needed for your particular experiment. For example, you may discover that certain markers don't need an FMO or that you need patient-specific FMOs for certain markers and not others etc. etc. etc. There are so many potential iterations here that the answer is the always flow answer of "it depends". But always start by testing them all for yourself and determine this empirically. (2) Yes using beads is fully acceptable. Just make sure they follow the same rules for compensation controls as if you were using cells.

@@ajarieger_flow Thank you so much for all the replies! I'm binging-watching all your series !! It has been very helpful in both reviewing the why for many practices we learn from other people but don`t really know why, and also getting some extra tips.

You’re not alone- many a matrix has been manually edited. Glad this helped!

Sorry I've gotten a little behind on my comments! Like any titration, you would want to look at all values (not just the high and low) and if I remember this experiment correctly, I think the best was 1:200

Sorry- I have gotten behind on my responses! In that case, because compensation doesn't care about biology (i.e. if your gates are in the right spot to capture the correct population) but only cares about the fluorochrome, you can just take the extremes of each side (the most negative and the most positive) and use that. Just make sure you have enough cells included!

You can add the count statistics when you create a table

Thanks for reaching out! This gets more challenging. I have generally first titrated the primary with a constant secondary concentration. I then pick 2-4 primary antibody concentrations that look the best and titrate the secondary antibody with each of those.

Be aware that non-specific staining will increase at high secondary antibody concentrations, so it’s important to compare the staining of cells with and without primary antibody present (secondary only control) at each concentration

@@ajarieger_flow thank you Dr.Rieger.

Thank you and glad you found it helpful! While it can be common practice to reuse beads to reuse compensation matrices, best practice is to run them fresh each time. Reuse puts you at high risk for compensation errors and artifacts in your data. So my recommendation is to run them each time with fresh controls.

@@ajarieger_flowI see. thanks a lot!

Hi! Sorry I've gotten behind on my replies. Generally in this case people will report %+ for AnxV+/PI-, AnxV+/PI+, AnxV-/PI+. If you are only interested in one of those populations then you would simply report that.

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