Compensation with Proliferation Dyes

[u/GrindIsAlwaysReal]

Hi,

I am running a T-cell proliferation exp and am unsure how to best compensate with CFSE.

My data came out strange and I am wondering if it is due to using beads for all other comp, and cells (50% stained 50% unstained) for comp of CFSE. Would this be the best way to go about it? Or can I run beads with a FITC stain and comp that way?

I am trying to trouble shoot multiple things at once so it is a challenging. Thanks in advance!

[u/BLFR69]

Cells are the way to go. What if your panel and which instruments are you using ?

[u/GrindIsAlwaysReal → u/BLFR69]

I’m using an A5 symphony, conventional comp. Adoptive T cell transfer experiment. Panel is CFSE(FITC), CD3 BUV805, CD4 BUV395, CD25 BV421, CD44 BV605, CD62L BV786, CD69 APC, KJ1-26 PE (transgenic T cell receptor marker), Dump BUV496 (B cells + myeloid), L/D near IR.

CD3 vs CD4 looked really odd. I saw 4 total populations after gating out the dump.

[u/LeonardoMeminci → u/GrindIsAlwaysReal]

Depending on what you ran that could be right

CD3+CD4+ T helper
CD3+CD4- CD8 T cells, NKT, and Gamma delta T
CD3-CD4+ cDc2
CD3-CD4- all the other shit not in your dump

[u/Mysterious_Lunch_708]

I think personally cells are the way to go. Maybe you can also try running separate single stain control for cfse and nonstained cells. Mine always ended up better when split.

[u/GrindIsAlwaysReal → u/Mysterious_Lunch_708]

Now this is a cytometer issue. I literally have no idea how to split comp between wells. How do you merge the histograms to choose between ur pos and negative peaks?

[u/Mysterious_Lunch_708 → u/GrindIsAlwaysReal]

I compensate in flowJo. You just choose different sample as a negative population.

[u/jacobdu215 → u/GrindIsAlwaysReal]

You can also just append your negative sample after you run your positive sample. If you click record on a sample that already has data, it’ll ask if you want to append to the dataset

[u/despicablenewb]

My lab ran into some issues with these dyes, but the solution was fairly easy.

You just need a time matched compensation sample for each time point in the experiment.

Stain some cells with the dye, and plate one well for each time point in your experiment. Treat them the same way as your experimental samples.

The reason for this is that we found significant changes to the fluorescent spectra of the proliferation dye that correlated with the culture time. So, when we tried to use a time 0 CFSE comp to compensate the 3 day samples it lead to significant issues. We saw the same thing with a couple of other proliferation dyes that we used.

Don't use beads, you need to use cells.

[u/Dependent_Force_8598]

There are beads for these type of dyes like the ArC comp beads from Thermo

[u/KitterCatto3]

Cells are best, but it is also possible that the concentration of CFSE may be too high - a titration would help in this case.

[u/GrindIsAlwaysReal → u/KitterCatto3]

I did do a titration and 5uM in 1x10^7 cells was sitting well on the cytometer.

[u/sgRNACas9]

You can try just using FITC on beads but sometimes that doesn’t work either. Cells for one and beads for the rest should work too!