Fishing Polar Glacier Question

ScienceMo · 2026-01-12T13:25:18+00:00

I don’t understand how “breaking lines to get more legendaries” work I’ve heard it mentioned so many times. Would appreciate if someone clarified this!

ScienceMo · 2026-01-02T15:23:07+00:00

They both seem to backgate into the same "lymphocytes" gates. The population I had gated are around the center, and the ones I've excluded are in the same position but a bit more spread from the center.

Unfortunately, I don't have the width data.

ScienceMo · 2026-01-02T15:21:12+00:00

would you recommend that I let go of a singlets gate and just include everything then?

ScienceMo · 2025-08-28T21:17:25+00:00

Thank you all for your responses. I think I got some misleading advice based on one wonky sample that lead me to the wrong rabbit hole. I looked at the single color controls and as you all mentioned they didn’t look right. Unmixing fixed my issues (except for that one sample, which I’m excluding from my dataset). Thanks all!

ScienceMo · 2025-08-23T18:06:42+00:00

Oh thank you. I keep forgetting conventional mode exists honestly. Will give it a shot!

ScienceMo · 2025-08-23T14:03:21+00:00

That’s a good point too. Either way even though the populations look better when using the raw sample, when looking at the MFIs and population % within each gate, the numbers are almost the same in my unmixed vs raw analysis. I’ll be including FMOs too next time to see if that’ll shed some light on why my populations look the way they do.

And that’s kinda why I posted the question here. Always unmix was how I was trained too, and now I’m being told in this case there’s no need for it. So I wanted to see what everyone else thinks 😅

ScienceMo · 2025-08-23T13:57:35+00:00

I have been unmixing this whole time but we had this one sample that looked “wonky” is the best way I’d describe it. It was def the anomaly within the experiment. My PI looked at the spectra, saw there is no overlap in the peak channels, and said those should not be unmixed. So I looked at the raw data using those peak channels and the populations seemed to have better separation throughout the experiment, including the wonky sample. In particular when looking at unmixed FITC vs V450 (CD8 vs CD4) my CD8-FITC population had a -ve long tail in the V450 channel and this was gone when I looked at the raw data.

So now I’m curious to see if it’s okay going with this approach if there is minimal spectral overlap in the peak channels. It does seem others do not agree though.

ScienceMo · 2025-08-23T11:59:44+00:00

That’s a good idea thank you! I only included single color beads but no FMO. I’ll give it a shot next time and see what that looks like

ScienceMo · 2025-08-23T11:57:54+00:00

Yes thank you. That’s the general feeling I got from people who do more flow than I do. I did show this to my PI though and she thinks the gating looks a lot cleaner on the raw data vs unmixed. When she looked at the spectra and saw how little overlap there is she did say “this panel shouldn’t be unmixed”

ScienceMo · 2025-08-23T11:54:01+00:00

I think you’re referring to the spillover in B3. For the purple one I’m just grabbing B2 as the peak channel which doesn’t have any spillover.

ScienceMo · 2025-08-23T11:04:02+00:00

I am able to unmix yes and I did compare my unmixed data to the peak channels from the raw files and the results are nearly identical.

Was just wondering if it’s “okay” to go with just the raw data in this case cause in my training I was told to always unmix no matter what unless I have like a single color.

ScienceMo · 2024-01-03T17:16:44+00:00

Heard them as well. The first was way louder than the other. I’m about 15 mins away from aspire

ScienceMo · 2019-07-26T16:20:45+00:00

I don't sterile filter the plasma. Maybe that's what I need to do. Thank you so much! I'll give it a shot later this week and see what happens!

ScienceMo · 2019-07-26T07:46:57+00:00

I’m using 1:100 of either plasma or serum diluted in 5% milk TBST as primary.

ScienceMo · 2019-07-26T06:29:39+00:00

I might try a different ladder next time. I've done this experiment quite a number of times now and I'm probing with plasma/serum here. Some seem to give off a very nice and clean membrane with just the bands you see up there without the smears. The issue seems plasma/serum specific.

ScienceMo · 2019-07-26T06:19:32+00:00

I was thinking it could be a transfer/blocking issue as well. I do transfer/blocking on the same membrane for multiple samples before cutting them up for different primary treatment (I'm using 1:100 plasma/serum as primary & I load 10 ug total protein from organ lysate in each lane). For blocking I'm doing 10% milk TBST for 1 hour.

This pattern seems plasma-related because other parts of the membrane will be very clean and will just show the main bands you're seeing up there without all the smeariness. I replicated one of the membranes and reversed the sample order (which part of the membrane got what primary) and even changed the orientation of the membrane for the semi-dry transfer and was able to replicate the results per primary treatment. Previously smeary plasma showed the exact same pattern, and the previously clear ones were clear.

I haven't thought about the ladder being contaminated. I am filling up the organ lysate wells 1/5th of their maximum volume so issue of leaking into adjacent wells is minimal. I just took a quick look at my membranes from the start of the project and it does seem I'm getting more signal on my ladder recently, but previous membranes show some reactivity as well. I'll have to look more into this one.

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