FlowJo [COMPENSATION]

[@prasanthchakrapani8105]

Thanks for sharing

[@rezammr843]

Perfect tutorial! Is it advisable to include a viability dye in the single-color control or it is better to have only single dye?

[@ajarieger_flow]

Thanks! And only single dye. If you add the viability dye, the signal from that will be counted as “overlap” in the channel where it gets detected ace subtracted out… and that’s no good!

[@rekhabt]

Thanks, Aja for this awesome video. I use whole blood staining of monocytes, I used to see double positive peaks in my compensation control for PE fluorochrome and the negative peak extends towards the negative scale. Can I still use the double peaks for compensation?

[@ajarieger_flow]

You can- compensation doesn’t care what the populations look like, just what the fluorochrome is. I would just gate on the higher peak (the brighter one) since this will best help follow the comp rules (as bright or brighter) and you won’t have a weird calculation of median from the 2 peaks.

[@worldwildmed]

Thank you for the helpful video. I ran a simple practice experiment where I stained with DAPI and a GFP-labeled ligand for uptake. I ran compenation but it seems like there is very little overlap (0.297 DAPI into FITC and 0.1006 DAPI into FITC) however my n x n plot looks linear, which I am interpreting as a positive correlation between GFP and DAPI. Shouldn't these look like two seperate populations with no relationship with one another after compensation? In fact it looks the same before and after compesation. I guess I expected no correlation between FITC and Pacific blue but I am not seeing that here.

[@ajarieger_flow]

A few questions for you: 1. Were the cells fixed (so all cells stain with DAPI) or unfixed (only dead cells have DAPI)? 2. In your GFP compensation control, what did the populations look like? (Clear negative/ positive; smear; all positive…) 3. Have you looked at your cells under a microscope?

[@emberthong7503]

I really appreciate it. It will be helpful!!

[@rezammr843]

I have one more question regarding the steps of compensation. Should I first set a gate on FSC-SSC, followed by singlet and doublet gating on all samples, and then transfer the single color controls to compensation section and proceed with compensation? Or does it not matter if I initiate the compensation process before the initial clean-up?

[@ajarieger_flow]

All comp programs will have you do FSC/SSC gating, which I would recommend. Doublets for comp don’t matter- all comp cares about is the fluorescence. And since you only have 1 colour in there, doublets would only be that colour anyway.

[@guhersaruhan-direskeneli2125]

Hello, I have a question about the compensated data that I have from the cytometer. As the flowcytometer only provides the compensated data, I do not know how to apply compensation in Flowjo, as the date is not "available" uncompensated.. Thank you for your comment in advance..

[@ajarieger_flow]

Thanks for your question! If your data is coming compensated off the instrument, you can tell this easily in FlowJo- the square beside your sample will have a grid in it if it is compensated (if the box is empty, the data is not compensated). As long as the grid is there, you are good to go! Even with the grid there, you can go into the compensation part of FlowJo (easiest way is to double click on the grid box) and look at the compensation matrix or redo the compensation in FlowJo. If you check out this video, it walks you through how to do it: kzbin.info/www/bejne/mIKZfYp3at1jnpI

[@arora1991]

Even after properly gating on the positive and negative peaks in the single stain control, can I still have issues with compensation in the samples? Instead of seeing cluster of populations I sometimes see slightly over or undercompensated cells. If there is over compensation, should I have a wider gate on the positive peak in single stain instead of gating just from the peak?

[@ajarieger_flow]

Yes this is possible (though without seeing the data, it is hard to comment on the potential source). Usually this comes down to the rules for compensation controls- go back and make sure all your controls follow all the rules.

[@alichamkalani6146]

Thanks for these great videos. I have a question: I stained isolated T cells with HLA-DR. However, the positive peak is almost on the unstained peak due to my cells not being activated. Since I will use this compensation for the next few days when the cells are activated, do you think it is reliable to use such compensation? Creating a tutorial for such cases is highly appreciated. Thanks!

[@ajarieger_flow]

Hi Ali- in this case you are likely to get compensation issues as your comp control will likely be dimmer than your samples (for more on this, I recommend checking out: kzbin.info/www/bejne/jpPEq6x_ntN5jKc and kzbin.info/www/bejne/iYnMi3-Fqc6XpsU ). In this case, I would recommend re-running this compensation sample with either activated cells or with compensation beads in order to get a sample bright enough to compensate your samples.

[@berkansavas5870]

Thank you Aja.

[@mervet643]

ı understand by means the video , As soon as use stain protocol without compansation from my samples and take my data from cytometer, I can compensation on flow jo. Isn't it? I asking because ı working with limited resourches in Turkiye. :((.

[@ajarieger_flow]

You can! As long as you have the appropriate single colour controls, you can do the compensation calculation in FlowJo if you didn’t do it in the cytometer.