It has been quite a while. I really do like your videos, very informative. As a junior immunologist, I have gained quite a perspective on flow cytometry thanks to you!
👍👍👍I have viewed this series several times WHEN I encounter some questions. I am now using Cytek full spectral flow cytemeter. Due to the high sensitivity of the machine, I need to be more careful on antibody titration. There is a question, that may be easy, but confused me a while. For instance, I titrate PerCP or other dyes, I also include live dead antibody, like Live/Dead Blue (a fix dilution, 1:1000), to exclude the dead cells to avoid autofluorescence. Here is the point, although PerCP is easy to be separated from live/dead due to the lower similarity index.Because I saw your titration vedio didn't include live/dead for each antibody titration. Just use size gating strategy to have live cells. I want to hear from you about this. Thanks in advance.
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!!
@@ajarieger_flow There is a question about the working flow of the Fortessa cytometer. For example, When I look at the spectrum viewer, I found laser 355 and laser 488 could excite fluorochromes, such as FITC, PE, and PE-cy7, though laser 488 could produce higher emissions of these three fluorochromes. I am wondering how the cytometer to avoid this issue.
@@ajarieger_flow There is another question about the compensation of fixable viability dye 506. The manufacturer recommends using live/dead cell mix to do compensation for that. However, my labmate told me a tricky way to use BV510 conjugated antibody instead of fixable viability dye for beads to do the compensation without using cells. Although I found the excitation and emission of fixable viability dye 506 and BV510 are almost the same in the spectrum viewer, it works for my work, I am still wondering is it good to do it? Because you said do not use different fluorochromes for compensation and samples.
@@feitu6403 This has to do with the laser delay and how the optical signals are captured. I would recommend taking a look at: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-5VM67E2DpL4.html
@@feitu6403 so while I have seen this done, I would highly recommend not doing this. Even if the peak is detected in the same channel, the spillover into different channels will be different, leading to potential compensation errors. Best practice is to always use the same fluorochrome to compensate as you have in your panel. If you do not want to use your cells to do this compensation, you can buy beads at bind the fixable viability dyes.
Hi Aja! Thanks for all your work. Your videos are helping me a lot and they are super detailed. I would like to know if you can make a video on how to analyze apoptosis. Thank you!
Hi Aja! Thank you so much for your FlowJo tutorials! I only discovered them today and already watched 3 episodes :-). I have a question, when we drag and drop our samples in FlowJo (or import them), the original names change to a number, which makes it quite difficult to know what sample belongs to what experimental condition. So far, we could not find a way to rename our samples in FlowJo. Do you have any idea/suggestion? Thanks a lot!
Thanks!! If there’s any topics you’d like covered, please let me know! What you’re seeing is usually a by-product of how the data was exported. When the names change to numbers this indicates that you exported the data out as the “experiment” FCS files as opposed to just the straight FCS files (there are 2 options in Diva- export FCS and export experiment; though both are FCS files, the export FCS ones are meant to be read everywhere while the experiment ones are meant to be read by Diva). I would recommend re-exporting as the export FCS version and your problem will be solved!
Hi Aja! Super clear and informative video thank you. I have a question... after applying the gates to all of your samples/groups, what if when you scroll through and notice that the gate needs to be adjusted and needs to be re-applied? I am having trouble doing this. After I fix the placement of my gate I wish to apply it to my samples again and its not allownig.. or is this not possible? thank you!!
Hi Melanie- thanks for reaching out! What I normally do is highlight my entire gating strategy on the sample that I changed and drag and drop this onto the group (in the top section of the workspace window). If this doesn't work for you, let me know!
Hi it was highly informative. I just want to know how we can compare number of counts in 2 different populations? I am using some particles and expecting that no. of particles will be less than the control. Thanks
Hi- sorry about the delay! I missed this one! Generally for GFP you would use an untransfected cell of the same type to see what the background intensity of the cell is
i use flowjo 10.0.1 version, for me some how "Create Batch report" does not work, any explanation why ? i follow your video accordingly to get that option done , however, the icons both (create batch report and separate pages) look dim
Thanks for tuning in! This usually happens when you have plots from more than 1 sample in the layout you want to batch from. If you go back and swap out plots so they’re all from the same sample you should be all set!
Thank you so much for this video! I did the same for the titration of CD4, but I have a question: why do I still see the negative population plotted in the final graph even after I gated only the positive one? Don't know what I'm doing wrong...Thanks a million
Thanks for reaching out! Will be hard to answer your question without seeing what you've done... if you want, you can send your files and workspace to me (aja@ualberta.ca)
Will do! I have a video I am currently editing that goes over concatenation (in the context of analyzing titration data) which can help you get started. I will get filming one on concatenation and tSNE. :)
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