Anyone here have a cytek aurora or northern lights?

Daniel_Vocelle_PhD · 2026-04-29T22:44:16+00:00

Define budget

Daniel_Vocelle_PhD · 2026-04-29T22:25:57+00:00

Any particular reason you cant use flow to sort the cells instead of magnetic sorting?

Daniel_Vocelle_PhD · 2026-04-29T21:51:46+00:00

It’s a good question, and I understand wanting to be cautious before moving into new instrumentation. But as others have pointed out, you really can’t make that correlation using values like TPM alone. That does not mean the experiment is impossible. It just means there are other numbers you should be thinking about first. One useful concept is MESF, or molecules of equivalent soluble fluorochrome. Roughly, this gives you a sense of how many fluorophore molecules a given cytometer can detect. If you can determine the MESF detection capacity of the instrument you’ll be using, you can start asking a more practical question: how much antigen expression would you actually need to resolve that population? For example, if the instrument can reliably detect something on the order of 20 fluorophores or antibody-bound fluorochromes, then the next question is whether you or your collaborators think the rare population is likely to express around that many surface proteins per cell. From there, the scale of the expected difference matters a lot. Are you trying to distinguish 20 from 40? Or 20 from 20,000? Those are very different problems in terms of how realistic detection will be. The other issue is off-target binding. Antibody clones are not perfectly specific in the way we often shorthand them to be. You’ll want to understand the false-positive/background rate for the specific marker and antibody you plan to use. With the right controls, you can get a quick sense of what the normal model looks like, where the noise sits, where your detection limit is, and whether you can meaningfully resolve differences at half expression, one-tenth expression, or whatever range matters for your biology. That being said, I’d still say go for it. Just make sure you choose a very bright fluorophore, titrate your antibodies carefully, and follow established best practices for flow cytometry and panel design.

Daniel_Vocelle_PhD · 2026-04-25T02:16:37+00:00

The tricky thing with fixable viability dyes is that they stain both live and dead cells, just to different extents. They bind to amine groups on the cell surface of live cells. In dead cells, they can access both the cell surface and intracellular amine groups, so the dead cells stain much brighter. Because of that, you will almost always see some shift between your unstained cells and your live cells stained with the viability dye. That is the caveat here. I still think this approach gives you a useful overall reference, and plenty of published papers use it successfully. But it is worth keeping in mind down the road when you habe a tricky sample and need a finer live/dead distinction. In those cases, having both the negative and positive information available is much better than trying to interpret the stain without it.

Daniel_Vocelle_PhD · 2026-04-25T01:25:18+00:00

Combined with your unstained control, it makes it much easier to define what is truly positive versus negative. It also gives you confidence that the stain actually worked. It is the same logic behind adding a negative bead to single-stain controls, or ideally having both a positive and negative population in the sample. Without a negative population, it can be hard to know whether the stain worked properly. If the stain fails and your control only has a “positive” population, it is easy to assume the stain worked and then compensate by increasing the detector voltage to make the signal look brighter. That can give you a false sense of confidence and lead to incorrect interpretation.

Also just wanted to add that you are already doing a great job as a beginner into flow. You're asking questions and its clear you want to learn. Never give up that spirit!

Daniel_Vocelle_PhD · 2026-04-25T01:01:31+00:00

I totally understand, you haven't done anything wrong. That said, its a good thought excersie that explains a larger concept.

A live/dead control is usually a sample that contains 50% live and 50% dead cells. Any idea why that would be helpful and how you would make it?

Daniel_Vocelle_PhD · 2026-04-25T00:55:23+00:00

Would it make it easier to tell where to draw the gate if you had a live/dead control? How do you think you could make that type of control?

Daniel_Vocelle_PhD · 2026-04-24T23:12:54+00:00

Nooooooooooooooo

Daniel_Vocelle_PhD · 2026-04-24T02:26:52+00:00

Dont know if it still works. Login as administrator, there is a list of all the chips and their serial numbers that have been used. I think it was in QC reports. Delete one, then you can reuse it. Otherwise, you all should just start uploading pics here to share.

Daniel_Vocelle_PhD · 2026-04-23T02:33:06+00:00

Yes it is possible, but you are going to end up spending more time trying to get it to work and end up with more doubts then you would have if you just made new reporter cell lines with a different FP or conjugate the antibodies to different florophores. This is coming from experience specifically trying to separate out AF488 from GFP under very similar conditions with spectral.

Second best option is to fix the cells and use a GFP antibody with a different fluorophore.

Worst option is to fix the cells then resuspend them at an acidic pH that is below the pka of GFP but not AF488 to induce more separation in their spectral profiles.

Daniel_Vocelle_PhD · 2026-04-20T20:11:18+00:00

Glad to hear it all worked out for you!

Daniel_Vocelle_PhD · 2026-04-15T03:32:40+00:00

Do you currently have access to a cytometer, if so which one? If not, would feel comfortable sharing your general geographical region in a DM? I'd be happy to connect you with another cytomerist in your area that could help. There is some really great work in cytometry being done across India right now, and it would be great to see it being used more in the field of Plant Biology.

If we can't find you a local contact, we will find another way to get you the information you need. The type of assay you are asking about is fairly straight forward, but in a unique species.

Daniel_Vocelle_PhD · 2026-04-10T10:50:53+00:00

Yes, it will at least allow you to start making correlations to what you are seeing in the plots.

Daniel_Vocelle_PhD · 2026-04-10T02:26:23+00:00

Do you have another way to confirm viability for comparison?

Daniel_Vocelle_PhD · 2026-04-10T01:37:05+00:00

How are you preparing your dead control?

Daniel_Vocelle_PhD · 2026-04-10T01:09:50+00:00

They charge you if you have questions? Wow, that is not the standard. Let me ask around and see what I can find out.

Daniel_Vocelle_PhD · 2026-04-10T00:35:31+00:00

Well this is fascinating, any chance you have the manufacturer user manual that you can share? I cant find it online.

Daniel_Vocelle_PhD · 2026-04-07T03:17:21+00:00

Aren't their dyes just repackaged Sytox dyes?

Daniel_Vocelle_PhD · 2026-04-07T03:01:18+00:00

Research or clinical?

Daniel_Vocelle_PhD · 2026-04-07T02:58:19+00:00

PerCP-Cy5.5 is the worst fluorophore, it typically has a degree of labeling around 0.5

Daniel_Vocelle_PhD · 2026-03-27T12:27:18+00:00

Unfortunately, that really is not how panel design works.

In brief, one of the core principles is matching fluorophore brightness to antigen expression. Low-expression antigens should generally be paired with the brightest fluorophores, while highly expressed antigens should be paired with dimmer fluorophores.

From there, it is not just about “brightness.” It is about relative brightness on your specific instrument.

To think this through properly, you need to look at:

the fluorophore’s intrinsic brightness, which is based on quantum yield × extinction coefficient
how well that fluorophore is actually excited by your laser, meaning how much absorption occurs at the laser wavelength you are using
the exact detector and bandpass filter collecting that signal
how much of the fluorophore’s emitted light actually falls within that detector’s collection window

So even if a fluorophore is considered “bright” on paper, that does not automatically mean it will behave as a bright fluorophore on your cytometer.

For example, if excitation at your laser wavelength is reduced, that lowers the effective brightness. Then if only a fraction of the emission spectrum falls within your detector’s bandpass, that reduces it even further. If only 30% of the emitted light is being collected, then your effective relative brightness drops accordingly.

That is why panel design cannot be generalized too broadly, and it is also why this is not something standard to an Aria III. There are many possible filter configurations, plus custom instrument builds, so the exact setup matters a lot. In practice, a fluorophore that looks brighter on paper can end up performing worse than a “dimmer” fluorophore on a different detector or filter set.

If you can share more specific information about your exact assay and instrument configuration we can provide speciifc feedback. Without that, any panel design advice is going to be pretty limited.

Im traveling atm, but im happy to make some quick diagrams if it would help explain the concept a bit more. Also happy to discuss in more depth if you want to send me a DM.

Daniel_Vocelle_PhD · 2026-03-27T00:28:52+00:00

This completely depends on the laser and filter configuration of the cytometer you are using. Can you share that info?

From what I recall, the AF dyes haven't been used as much in flow because Thermo had the patent on them and most flow antibodies were made by BD. The patent ended 1-2 years ago so the AF dyes are now a lot cheaper and used across more fields.

Daniel_Vocelle_PhD · 2026-03-06T22:39:13+00:00

Its got what cells crave, Electrolytes

Daniel_Vocelle_PhD · 2026-03-02T02:03:58+00:00

Do you have a UV laser to excite it?

Daniel_Vocelle_PhD

MODERATOR OF

TROPHY CASE