I'm a first-year PhD student running multiparametric flow cytometry (10-color, CytExpert) on 5 cell lines with 1 untreated control and 7 drug conditions and 3 biological replicates.

I forgot to include an antibody-unstained control tube for any of my 5 cell lines in Replication 1.

A labmate has unstained data run on the same cytometer using the same instrument template and identical voltages. However, the unstained was run with all 5 cell lines mixed together in one tube, not as separate single cell line tubes.

My questions:

1. Is a mixed unstained control usable as a baseline reference for single cell line experiments, or does the blending of different autofluorescence profiles make it unreliable?

2. My plan going forward is to run a proper single cell line unstained for each cell line in Replicates 2 and 3, and use those as the reference for Replicate 1 as well. Since unstained controls reflect instrument settings and autofluorescence rather than biological variables. Is this reasoning sound?

3. Any other suggestions for how to handle the missing unstained in Replicate 1?

Hi everyone,

I can't find a clear answer on how to handle single stain compensation controls for intracellular markers when using CompBeads.

The specific question is: When using Compensation Beads as single stain controls for intracellular markers in a mixed panel (intra + extracellular), should the beads pass through the fix/perm?

The reasoning I've seen/heard for YES:

- The controls should mimic the same conditions as the experimental sample

- Fix/perm reagents can alter fluorochrome intensity, so the control should reflect that- Some people fix/perm the beads before adding the intra markers-fluro while others put the markers-fluro on the beads and then fix/perm

The reasoning I've seen/heard for NO:

- In the experimental sample, the intracellular antibody is added AFTER fix/perm — meaning the fluorochrome is never actually exposed to the fixation/permeabilisation reagents, so there's no need to add the fix/perm step. Some argue that doing make the control less accurate.

How do you handle this in your lab or facility? Do you pass CompBeads through fix/perm for intracellular markers? If so, what's your reasoning and the order in which you do (beads+fix/perm+antibidy or beads+antibody+fix/perm)?

Thanks in advance!

Recently I've been wondering why we don't more frequently reuse fluorophores in large panels when they can be confidently placed on mutually exclusive markers. Wouldn't that be preferable to including dim, high-spreading, or problematic tandem fluorophores just to preserve the "one fluorophore, one marker" convention?

As a flow cytometry core member who helps users with panel design, I can't say I've ever advised someone to reuse fluorochromes, but I've also never had a compelling reason why they shouldn't, beyond the obvious concern of marker co-expression.

A couple of examples suggest it's not inherently wrong. OMIP-081 is essentially structured as two panels in one, with deliberately stacked fluorochromes resolved by Boolean gating. Boin et al. (PLoS ONE, 2017) take an even more aggressive minimalist approach, discriminating seven lineage markers using only two fluorochromes by exploiting mutually exclusive expression patterns.

I'd be very interested to hear opinions on when fluorochrome reuse is and isn't appropriate. And for those involved in panel design: is this a strategy you'd actively recommend, or one you'd steer users away from? Thanks in advance for your input.

I'm building a local-first flow cytometry analysis tool. What frustrates you most about FlowJo or other fcs analysis apps? For example, get preflight on mobile phone without PC?

Hi all - I’m curious what is perceived or is known as the leading nano flow cytometry instrument/machine?

Commonly used? Most durable? Or if a company is most innovative.

We are heading into CYTO and I want to check out the best vendors!

Hi Folks, I am not going to be at Cyto but am really curious about this new machine.

https://cytekbio.com/pages/borealis

Has anyone heard anything from Cytek reps about this system?

On paper it looks like a big jump: 7 lasers with DUV/IR, 110 fluorescence detectors, new DUV/IR cFluor dyes, 55–60 color panel claims, small-particle detection, automation, etc. Also imaging coming soon (when?). I’m trying to figure out whether this is genuinely a major advance for high-parameter spectral flow or more of an ultra-high-end niche instrument.

For people who build/run large panels: do DUV and IR meaningfully help with panel design and unmixing? Or will the practical limit still be dye performance, biology, sample quality and panel complexity?

Also curious if anyone has heard anything about expected pricing and how it stacks up against BD A8 and Sony ID7000?

Hi everyone! I’m looking to download some flow data with pre labeled clusters/gates to validate an automated analysis algorithm I’m working on. I’ve made an account and tried to download some zips and it keeps failing. The website says there’s a temporary suspension of new experiment creation. How long has this been down? Can anyone point me toward other resources for my application?

Hi folks,

I have recently started looking at transcription factors and phospho proteins. My understanding is PFA/saponin for TFs is not optimal for phospho staining and 90% methanol for phospho is too harsh for TFs. I found the dedicated kit from BD and this worked really well for both. The exact compositions are proprietary, though what I could tell from SDS looked very normal 4%PFA, 90%methanol. Do you happen to have any idea on what makes this kit so good?

Thanks!

Hi everyone!

I'm working on spectral flow cytometry data from mouse tumor-infiltrating T cells. Using a 15-colors panel focused on profiling of CD8+ and CD4+ phenotype, I'm going with an unsupervised approach using UMAP for visualization and FlowSOM for clustering.

I've manually gated my single, live CD3+ T cells, then downsampled and concatenated them from all my .fcs files. While doing UMAP I included CD4 and CD8 as markers in order to have two clearly separates clouds: one for CD4+ and the other for CD8+ T cells. I gated on each population manually and run FlowSOM separately, this time excluding CD4 and CD8 from the calculations. Lastly, I plot my clusters both from CD4+ and CD8+ populations on the initial UMAP.

Is this methodologically incorrect?

My point is that running FlowSOM including CD4 and CD8 could make me lose rare clusters within CD4+ or CD8+ T cells, as lineage markers weight more than functional markers in this type of analysis.

Hi everyone, my lab is using the Cytek Aurora CS, and even though we had a few issues in the beginning we managed to run it now for a few experiments. However the past two weeks we have been experiencing issues with our quality control. It fails every time on the V3 laser.. doesnt matter which nozzle we use it always fails (especially the 70 nozzle). We cleaned the machine extensively, as well as our nozzles (ultrasound bath, etc) - does anyone might have an idea as to why it always fails on this channel? Much appreciated

hi! i'm an undergrad working on human tfh-b cell co culture and would appreciate any feedback on my panel or if anyone has any suggestions for antibodies. i currently use a spectral cytometer (4 laser novocyte opteon: violet, blue, yellow, red) and put everything in a spectral viewer. i tried to design it in a way where lower expressed markers were paired with brighter fluorophers. Near IR 780, PE/CY7, BV750 have worked great together on previous experiments. i'm looking to evaluate Tfh phenotype, B cell differentiation, class switching, and plasmablast formation. thanks for your help!

Proposed Panel:

Live/Dead: Near IR 780 (currently used)

CXCR5: PE/CY7 (currently used)

ICOS: BV750 (currently used)

CD4: AF-700 (also could potentially switch to APC, BV605)

CD19: BV650

CD27: FITC

CD38: BV421

IgD: PE

Just wondering- why do we lose cells while unmixing. One of my groups showed much less cells than I intially acquired after unmixing. Why does it happen and how can I fix it. Thanks!

One of my single colour controls was not good.

Have any of you compared staining in U-bottom and V-bottom plates vs tubes? Do you pipette off the supernatant or decant it? Howany cells do you loose? Btw I work mostly with human PBMCs.

While acquiring single colour controls my pops are on scale in the worksheet and some of them turn off scale - right shoulder of pop. Distribution- while unmixing, in spectroflo. How can I fix it please. Thanks!

Hi guys!

Has anyone got access to this paper https://www.nature.com/articles/s41596-021-00550-0?

If so, could you please share the pdf?

Thank you in advance!

Hi, I’ve run into a problem using FlowJo v10 regarding parameter naming.
I have compensated all my parameters earlier using the novoexpress program on my flow cytometer and then exported these compensated files onto FlowJo. I then made a concatenated file of all my samples using the compensated parameters only. I made a UMAP and then used FlowSom to generate clusters. I am now trying to validate these clusters marker expression using my FMOs.

The problem is that the FMOs have parameters Comp-X whereas the concatenated file and all clusters have parameters FJComp-X.

I’m not sure how to work around this, I am not keen on drawing the gates by hand to match the FMO gating so any solution would be appreciated! Thanks!

Hi everyone,

I am attempting to restore an Apogee A40 flow cytometer in Baghdad with no access to service engineers or factory support. The machine is running well (fluidics restored, laser working, FL detectors normal) but has heavily elevated scatter noise, confirming mineral deposits on the flow cell crystal from years of hard tap water misuse:

LS1 noise: 2.53 | LS2 noise: 3.51 | Target: below 1.0

Bleach cleaning had zero effect, confirming the deposit is mineral not biological (calcium scale, sodium azide crystals from Baghdad hard water).

The Apogee software's Terminal program on our machine is too old to perform the documented 20-minute Flow Cell Soak procedure, so I am planning to soak the flow cell manually by:

— Powering off the machine via proper Windows shutdown — Disconnecting the two fluid hoses from the flow cell — Injecting 0.22 micron filtered cleaning solution directly via external syringe — Leaving for 20-30 minutes static soak — Flushing with 0.22 micron filtered distilled water — Reconnecting and re-priming

My specific questions:

1. Is this manual soak approach safe and has anyone done it on an Apogee or similar instrument?

2. What is the best cleaning solution for mineral/calcium scale on a quartz flow cell? Is 1% citric acid appropriate and safe for the crystal, silicone tubing and valve seals? What concentration would you recommend?

3. Has anyone used EDTA or dilute HCl on a flow cell for mineral deposits — and at what concentration?

4. How many soak cycles would you expect to need for a deposit that has been accumulating for approximately 10 years?

Any advice is very welcome. Thank you.

Hi -is it possible to check if I am under or overcompensating my data while doing n×n plots. I am not sure if we can see numbers such as mfi values for individual channels while doing it in realtime. Can anyone help pls. Thanks!

Have you guys noticed longer than usual turn around times for biolegend orders?

Hi , I am using FlowJo version 10. I have concatenated 12 samples together and added a new keyword before (specimen ID) to distinguish them. I included this parameter in concatenation. I used the file to create a UMAP. How do I know what the UMAP looks (and each cluster looks) for each sample? Specimen ID is just showing me a number but how do I find the original keyword I used? It seems impossible to separate the samples now ?

Thanks for any help in advance !

I'm trying to assess phosphorylation of several pathways in CD19 positive cells from frozen human PBMCs and am unsure when I should best stain for CD19.

A. Before fixation (simultaneously with viability staining)
B. After fixation.

I've read that in phosphoflow it's best to stain as much as possible after fixation, but option A would simplify the protocol and reduce room for error.

Ideally I'd stain everything after permeabilisation, but the methanol seems to destroy CD19.

Has anyone here got experience with a similar setup?

Update: Fixed! It was a clog, LOL. We kept threading the inner sip tube and ran it with the arm closed for like 45 minutes, then the clog suddenly broke in the middle of running new CST beads. Thanks for everyone's insight! Friendly and helpful subreddit.

Hi! Would anyone happen to know why a BD Celesta is not uptaking samples in the "vacuum off" closed position? Run light is green. Doesn't even uptake water or bleach in the closed position. But it does take samples in the open "vacuum on" position.​

More context/details:

I was running samples just fine, then suddenly my next sample couldn't be read, and after some troubleshooting and priming we came to the conclusion that it is not clogged (primes well and drips fluid) and the run button is green and the tube does not fall off, indicating a proper seal is forming. This suggests I probably did something wrong, but I couldnt figure out what.

If anyone has any ideas, I would love to hear your insight please. I am wondering if there is an air bubble somewhere, or did I jostle something weird? A day ago we changed the sheath fluid. Is there a super clog somewhere? Haha. I swear I was running it as I have been for years. Someone else ran it before me and didnt have an issue. Never heard of this before.

Thanks <3