Thanks! There won't be such a long break until the nest one :)

If there’s any topics you would like covered please let me know!

Thanks! Anything you would like covered, feel free to reach out!

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!!

Thank you! And yes- I will get that done for you :) Annexin V/PI apoptotisis assay?

@@ajarieger_flow Yes, Annexin V/ PI .Thank you!

Congrats! Enjoy the PhD road

Thank you! Let me know if there are any specific operations you would like more information on :)

@@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: kzfaq.info/get/bejne/a7x9aZp2l6rUfGg.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.

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!

Otherwise you get into editing keywords… I would stay away from that

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

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!

@@ajarieger_flow thank you so much it worked! :)

Thanks!

You can add the count statistics when you create a table

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!

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)

@@ajarieger_flow Thank you so much I will do it as soon as possible.

I don’t have any lectures specifically for this… but the intro to flow playlist and the sample prep playlist will be helpful!

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. :)

@@ajarieger_flow Awesome! Thank you very much!!

@@ajarieger_flow awesome thank u!

@@ajarieger_flow thank you so much 🙏 you are the best!

@@user-tb6tj9tt1y tSNE video will be coming out next week- stay tuned! I have not forgotten :)