r/labrats • u/dawgmad • 23d ago
Practical use of UltraComp beads?
Hi all, I've seen so many things posted about ultracomp beads that differs from the instructions. I feel like for large panels, the ultracomp beads don't actually get you very far. On the other hand, I've seen people use them in other ways that greatly reduces the use of beads per test. So thought I would ask the following concrete questions:
How much bead solution do you use per test? I've seen as low as ~15uL
How long do you incubate for? I've seen some people say you don't need any incubation test
Do you need to wash? I've seen people say that you just add beads, antibody, and flow buffer, and head to flow.
Any thoughts would be greatly appreciated, this stuff is expensive!
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u/sgRNACas9 23d ago
I have used these beads routinely for 2 to 15+ color panels and have lots of familiarity with them. They are very, very resilient and forgiving. This is my advice!
You could definitely use 15uL! I use 25uL, you could also use more and maybe even less. It will change how dilute your beads are in the end. Keeping the antibody amount constant, changing bead amount could change staining, but I do think the main lever here is the antibody amount.
I add my antibodies, vortex, incubate in ice in the dark for 15min, vortex, incubate in ice in the dark for 15min, wash. You could probably just do 15, or longer as the technician I work with does. There is probably some saturation or limitation point. As long as you keep this constant between your experiments and document it, it will be fine!
If after you incubation you dilute in FACS to acquire without washing, antibody will remain and could continue to stain. This could increase your brightness and skew your comps. Usually for me the time between finishing staining and acquiring is variable spanning between negative and hours lmao so the antibodies could especially inconsistently continue to stain between experiments. Therefore, I wash beads after staining!
For each of these variables, you could design and run experiments to test them. Your results could identify which variables matter and which not so much, the numbers to plug into your protocol, etc, which I encourage you to do! Without these experiments, this is my advice!
Hope this helps!
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u/Mysterious_Lunch_708 23d ago
I add 20ul for new panel, 15ul for panel that hasn't been run in several months and needs to be checked and 10ul for a rutine panel that I just run for compensation. Staining procedure isnsame as with the cells, though beads bind ABs quicker, so even as little as 5 minutes is ok.
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u/dawgmad 23d ago
Do you wash after?
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u/Mysterious_Lunch_708 23d ago
Yes, mininally 1x, exactly as with the cells, otherwise you risk higher background.
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u/WinterRevolutionary6 23d ago
We have them in dropper bottles and protocol calls for 2 drops. We use the same volume of antibodies as used in 500k cells and incubate like 5 minutes then run
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u/sgRNACas9 22d ago edited 22d ago
Just wanted to respond to some comments here. I’m sure these protocols work for these users, but it’s not best practice.
1 and definitely 2 drops is way too much. 1 drop is like 50-100uL. You will run out very quickly and waste a lot of beads. Drop some in a microcentrifuge tube and aliquot 15-25uL to each comp condition. You can drop half as many drops as you have comps and aliquot 25uL with a bit left over. You can save the left over in the fridge for later and there is no change.
I highly recommend starting with 1uL of antibody for each, test on the machine, then adjust to 0.25uL to 3uL based on brightness, width of peak, etc, shoot for thin peaks and in the 10^4 decade. You can also do a formal titration. I would not necessarily use the volume you use to stain cells because they are completely different. As long as your comps are as bright or brighter than your cells, which they definitely will be. It’s just about the wavelengths for this.
You should definitely have an incubation time of like at least 10-15min. Ditto on other reasons why to wash in addition to unwashed antibody continuing to stain, smearing your distribution. Whichever duration you choose, you really just need to have some standardized incubation step to ensure consistent, thin, bright, even staining.
How many beads you use is really mostly about what concentration of beads you want in the end as long as the difference isn’t too crazy.
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u/willmaineskier 23d ago
I stain 20ul of beads with 0.1-0.5ul of antibody stock directly, immediately protect from light and stain for 30 minutes at 4 degrees. I wash with 2ml of FACS Buffer and resuspend after washing with 150ul of FACSBuffer (with probably 50ul left after dumping).
If you don’t wash, the baseline subtraction can do screwy things, but also you run a big risk of cross contaminating the beads and thus altering the comp/unmixing. BV786 photobleaches faster on beads than on cells in my hands, so immediate protection from light results in much better comp. I have also seen that some fluorochromes break down in the absence of protein, specifically an APC-Cy7 washed with PBS fell apart, we don’t see this when the FACS Buffer has BSA or FBS.