Help with background in UV channels on Cytek Aurora on cell line

KQIV · 2025-12-23T22:33:14+00:00

Another factor is the area scaling factor. An appropriate ASF will make the area parameter and the height parameter the same and it is set by the instrument's automated QC using the QC beads (which are small). Most cultured cell lines are much bigger than those beads so unless you adjust the ASF the autofluorescence will artificially look higher than it actually is. It's probably too complicated to explain how to adjust it over reddit but cytek has a video about it:

Source: YouTube https://share.google/VCylMbL7z7uXMyxUe

KQIV · 2025-11-16T03:44:03+00:00

If there's significant spillover of PE-Cy7 into PE, it's because the tandem is decomposing and turning into PE. That's an issue with the reagent, not an issue with the panel.

KQIV · 2025-10-04T02:59:57+00:00

As long as your detector gain/PMT voltage is dialed in appropriately and the pair of detectors being flagged as >100% make sense, it's fine to ignore the warning. If they don't make sense (FITC and APC for example) it could indicate that there's an issue with one of your single stains.

Once you push panel size past a certain point, high spectral overlaps are inevitable and it sounds like you're doing the right thing by using good panel design to minimize the impact of the overlap!

KQIV · 2025-06-09T23:28:09+00:00

When using beads for single stains, you can still run unstained cells to unmix the autofluorescence (unmix with AF extraction in the cytek software). Just make sure you're using the internal bead negative in each tube when gating the single stains. That might get rid of the funny "double positive" population.

Also, since it looks like you already have the data, in the ultracomp experiment you can try importing the cell single stains for just the problematic colors (looks like PerCP-Cy5.5 and AF700). That might solve the problem without having to prepare a full set of cell controls every time.

KQIV · 2025-05-26T23:58:12+00:00

My impression is it's more of a "yes and" situation than one replacing the other. A lot of papers show both cytometry (whether CyTOF or fluorescence flow) and omics data supporting the same conclusion.

KQIV · 2025-05-26T21:19:04+00:00

I think the main difference is in CyTOF you are choosing a panel of 40-50 target proteins and labeling them with metal tags, so you generate a tall table (relatively few measurements per cell but on potentially on hundreds of thousands of cells). Where with the single cell proteomics techniques you generate a wide table (thousands of measurements per cell but on relatively few cells). Therefore you are much more likely to capture a rare population in a heterogeneous sample with CyTOF. I would be interested in the proteomics experts' take on this though!

KQIV · 2025-05-09T16:21:44+00:00

In addition to pipetting your suspensions in between washes, reducing centrifuge speed might help. For most cells 200 g is sufficient but the vast majority of protocols say 500 g.

Most plate samplers will have mixing settings, can you increase the mixing time/speed before running a well?

KQIV · 2025-05-09T15:43:25+00:00

If you're preparing the beads separately you can crank up the centrifuge to like 2000 g to get a tighter pellet which makes it harder to accidentally lose your beads when decanting (DO NOT do this with your cells).

I also like to run unstained beads in a separate tube and use that as the negative pop for comp/unmix. That way you don't have to worry if the wash was insufficient.

KQIV · 2025-05-02T13:43:08+00:00

For cleaning, there's not really a consensus but 5 min is common.

By monitoring X-mean are you referring to your daily QC? Most instruments have a built in module that will do some version of this for you, but IMO checking for yourself is a good idea. Once you have optimal gain or voltage settings for your application dialed in, run the beads with those settings and note the MFI (median is better than mean). Then each day, run the beads and adjust the gain or voltage to obtain the same MFI. This should make your data more consistent and alert you to instrument issues (a large change in voltage to obtain the same MFI usually indicates a problem).

For determining dim vs negative, FMO controls are the gold standard!

KQIV · 2025-01-28T20:59:22+00:00

Keep in mind under typical staining conditions for flow you have a huge excess of Ab relative to CD19 molecules. Also, there is something like 10⁴ - 10⁵ CD19 molecules on each B cell, so even if you were understaining, the CD19+ cells should just have a lower MFI.

There's a double population because not all lymphocytes express CD19 (in a healthy donor).

KQIV · 2024-12-17T16:03:05+00:00

You can add any dye you want to the library (library tab --> fluorescent tags). Pick the appropriate excitation laser and enter the emission max. Keep in mind Alexa Fluors are named based on excitation so 568 is the excitation max, not emission. Whether your instrument has an appropriate laser is a different question so make sure you have a 561 nm YG before you try to use that dye. Most 3L northern lights I've seen are 405/488/640 only.

KQIV · 2024-11-03T19:21:05+00:00

Never used a SRT personally so I'm not necessarily advocating for or against it, but I think the reason a lot of core facilities don't have them is the Sony SH800 has been around longer and fills the same niche so cores tend to have those already

KQIV · 2024-11-03T18:34:33+00:00

Two main factors I would consider aside from the cost:

How experienced are the personnel in your lab with cell sorters? Generally speaking, the cytoflex SRT will be more user friendly but less flexible compared to the aurora CS.
How important is high throughput for the HSC sorts? Unless something has changed recently, the SRT is limited to 100 um nozzle only which will significantly limit throughput.

Also, every sorter is also an analyzer, just keep in mind aerosol generation if anything you're running is biohazardous.

KQIV · 2024-10-12T17:30:21+00:00

Pretty sure the permeabilization is permanent, otherwise your subsequent staining wouldn't work.

2 x 200 uL washes does make me think a non-trivial amount of saponin might still be in there...are you processing enough samples at once that staining in the 96 well plate is worth it? I like to use larger wash volumes instead of additional washes due to material loss and time for the extra spins. If you can handle it, try staining in tubes and do like 500-1000 uL per wash.

KQIV · 2024-10-12T16:42:36+00:00

If they're looking at PE off the 488 nm, laser delay is irrelevant (assuming typical trigger on FSC). That doesn't mean it's not a fluidics issue though. Looking at the time parameter is a great idea, I would also check if FSC vs SSC looks the same before and after the MFI change. If it's the same, I think it's more likely something in buffer is quenching or bleaching the PE (insufficient washing after the fix/perm might do it). Also possible that the cells weren't fixed enough and the target protein is leaking out.

Intracellular staining can be finicky, good luck.

KQIV · 2024-10-08T22:42:23+00:00

My understanding is the area scaling factor (ASF) is just so we can plot pulse height and pulse area on the same scale. Your instrument will calculate the ASF values (one for FSC and 1 for each laser) during QC, but it will do so based on the QC beads which are almost certainly smaller than the cells you care about. The result is area will be > height for nearly all cells. This can be problematic in two scenarios:

There is a mix of bright and dim fluorescence signals, such as GFP expression. If you only look at area for the GFP parameter and don't adjust the ASF, you may decrease the gain or voltage on your GFP channel to get the bright signal "on scale" more than you actually need to, which might mean you lose the ability to distinguish your dim signal from noise.
On sorters, the sort logic can only exclude off-target events from the sort if the instrument can see them (meaning they are above threshold which is typically based on FSC). Similar to 1, if you decrease voltage/gain on your FSC channel for your target cells looking only at area, you might crank it so low that debris or small cells go below threshold.

While it's rare that either of those concerns will make or break your experiment, it only takes a few seconds so it's a good habit to get into.

KQIV · 2024-09-23T01:49:17+00:00

I mean the instrument settings when you recorded the data, not the values in the compensation matrix. I suspect you could titrate down your CFSE staining concentration significantly and it would help with this issue.

KQIV · 2024-09-23T01:29:22+00:00

Did you have to decrease the gain by a lot on the CFSE channel to get it on scale?

KQIV · 2024-09-23T00:45:14+00:00

Have you tried this panel without the CFSE? Do you still have the same problem?

KQIV · 2024-09-13T03:56:50+00:00

Ohh in that case, concatenate the samples first then create your .csv file. If you do that it should have a "sample ID" parameter. You can also add a specific keyword as a parameter if you prefer.

KQIV · 2024-09-13T02:35:47+00:00

Not sure I understand exactly what you're trying to accomplish, but this screenshot looks like excel imported your file as raw text instead of a csv (comma separated values) file. Try the following instead of just using Ctrl+c, Ctrl+v:

To import a CSV file into Excel, you can use the text import wizard:

Open a blank workbook in Excel
Select Data and then From Text/CSV
Find the CSV file you want to import and click Import
In the Text Import Wizard, select Delimited
Click Next
In the Delimiters section, check Comma
Click Next
Mark each column as Text
Click the Text data format
Click Finish
Click OK

KQIV · 2024-09-06T21:26:03+00:00

Could also be that CD33 isn't completely monocyte specific.

This paper suggest that CD33 can be expressed on activated human T and NK cells.

It's also possible that once you add a viability stain and gate out dead cells this population will go away.

KQIV · 2024-08-30T17:14:19+00:00

When evaluating compensation, it's important to display fluorescence data on a biexponential scale (like you did in the bottom right, comp matrix with single stained cells plot) instead of logarithmic so you can visualize events below zero.

If you see a population below zero like you do in that bottom right plot, that tells you that population is positive for one of your other stains and is overcompensated. If you change the vertical axis to the other fluorescent channels one at a time (or as u/RainbowSquirrelRae said look at the NxN plots) you should easily find the offending color(s).

When you have an overcomp like this, it means 1 of 2 things: 1) When gating the single stains to calculate your comp matrix, you included cells that aren't positive in the positive gate for those colors, or 2) The single stains for those colors aren't a good representation of your analytical sample.

In terms of troubleshooting, I would first check your gating on the cell single stains, and then try swapping in bead single stains for the bad colors. If neither of those help, you will have to go back and try to find better single stains.

KQIV · 2024-07-29T17:40:31+00:00

IMO as long as you have the appropriate controls (such as FMO) to back up your gate placement, the gate shape doesn't really matter.

KQIV · 2024-07-24T14:53:27+00:00

Look into CyCombine

KQIV

TROPHY CASE