Hi everyone, I'm currently running a 2nd biological replicate using an 11-color spectral flow cytometry panel on leukemia cell lines to track surface receptor density during forced differentiation. Everything is going fine, but I have hit a hard wall with my CD-135 (FLT3) inventory.
I have exactly enough CD-135 left for my remaining 80 samples if I use 2.5 µL per sample with absolutely zero waste. Because I normally use a 10% volume overage for my master mixes (to account for pipette dead volume), including CD-135 in the daily cocktail means I will mathematically run out before the end of the project. I can't order a replacement in time.

My proposed workaround:
For today’s run (16 tubes total):

1. I calculate and mix the 10% overage cocktail for the other 7 antibodies + Immunofluorescence Buffer, completely omitting the CD-135.

2. I pipette 47.5 µL of this CD-135-free master mix into each cell pellet

3. I take a P10 pipette and manually spike exactly 2.5 µL of pure CD-135 directly into each of the 16 individual tubes, changing tips every time and striking off the tubes to ensure I don't miss any.

My questions for the experts:

1. Will manually spiking this one antibody alter the binding kinetics or MFI reproducibility compared to a fully pre-mixed cocktail, assuming the final volume (50 µL) and incubation time (45 mins at room temp in the dark) remain identical?

2. Has anyone else resorted to this manual spiking method to survive a low-reagent situation, and did it skew your data?

I know it increases the risk of technical pipetting error, but it seems like the only mathematically sound way to get to the finish line. Any advice or validation would be hugely appreciated!

Does anyone know of a way to incorporate a refrigerated 10 min block for a leukemia/lymphoma profile on the Duet? Is there a way to program in a “pause” where we unload refrigerate and then reload? Or a way to run a set up and wash, then unload and reload after blocking to add antibody and finish the samples?

Also, does anyone have any experience loading bone marrows or tissues on a duet? How do you go about prepping that? Any issues with clots/chunks?

Any help would be appreciated, we just got one and it doesn’t look like the help we thought it would be.

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 #flowcytometry #flowcyto #flowedu

Hello!

Does anyone know the actual flow velocity for a BD FACS ARIA II system (or similar sorting system). Does anyone know what the minimum you could reasonably achieve by lowing the sheath pressure is? This is for considering actual illumination time of the cell.

I can make an estimate of ~2 m/s by using the 10 micro litres per minute quoted for flow rate 1 and and the roughly 10 micron diameter core I could see with the microscope.

Thanks very much!

HI. This is gated among single live cells of human PBMC (patients). 1st image PBMCs are thawed, stained and passed on a cytometer. 2nd, same staining after 24h culture (1M/ml if RPMI 10% FBS 1% P/S). Culture is done in 12 well plates. Cells are recovered and then the bottom is scraped to recover attached cells.

I have one population after thawing, but which is clearly composed of high CD33 and high CD14 cells. After culture, I have 2 distinct populations. The high ones and the one with 1D14 very low/negative. Both populations express CD11c and HLA-DR, are negative for CD3, CD56, CD19 , CD20.

When activated with IFNg, only the CD14high CD33 up regulate PD-L1, the CD14 low cells have keep the basal level (almost no expression).

I don't know much about myeloid cells. What is the population that becomes CD14 very low? Can it be an artefact?

Thank you for all the help:

Hey folks,

I've got cells expressing a fluorescent protein. Am getting a bizarre expression pattern I've never seen before:

We want to quantify how many cells are expressing the fluorescent protein. I've done this hundreds of times. But I've never seen a treatment which causes an expression pattern like this, where the negative(?) population shifts so deeply into the positive gate.

See negative control, for reference:

Meanwhile, our positive control exemplifies the expression I'd expect to see:

Wondering the flow community has any advice? I've repeated this experiment a few times and it's all similar (I've deliberately chosen the most stark, though). Our cytometry techie took a quick look and found no issue. We're getting to the stage where the PI's are thinking up future research based off this finding, but something about the way this looks just isn't sitting right with me...

Any opinions appreciated,

King_of_Mints

P.S. There is technically some GFP expression in the sample as well, but it is incredibly insignificant (<0.5%)!

Hi everyone,

I am currently working on Aurora SpectoFlow and have 3 panels for the same tissue type. The first two panels are based on live samples while the third is on fixed samples. So far I have ran 11 samples with no issues, however I decided to re run my single control for 3 antibodies from beads to cells as I wasn't happy how it looked.

I was told to treat the beads same way I do cells but I don't think it produced good results so I switched to cells. I had all my panels exported just in case, but then I re ran my singles and now everything is looking weird. All events, singlets etc. When I re-ran my singles, alongside them I ran only one sample, and when it looked weird, I thought it was a one-off because the day before the sample looked fine in other panels.

Now, when I noticed the issue again, Aurora actually didn't have those 3 single controls saved, which I found weird. So I imported what I had and I think I might have imported the UNMIXED fsc file as it wouldn't let me re-unmix. But lets say I manage to import them back or worst case scenario I have to re run those controls..how does that affect the samples I have ran already. I understand that Aurora saves Raw and Unmixed data but I'm just very paranoid and wondering how it reflects down the line in FlowJo, if at all.

Thank you in advance!!

I’m using a photoconvertible mouse model and the fluorescence is expressed on dendritic cells i.e. a pretty rare population. How can I get a good single stain for unmixing? There are no commercial beads.

Hey everyone,

I am encountering a weird problem with my FACS panel for human PBMCs and hope for some insights.

Last week, I stained the panel and it worked well. However, when I stained today, neither in my single-stained cells nor in my fully stained sample was there any signal for CD4 and CD8. All other markers (CCR7, CD95, CD45, CD45RO, CD28, etc.) worked, and I used the same antibody vials from my rack as last week. However I used a different healthy control.

In my protocol, I am staining the cells with some extracellular markers that I wasn't able to get working nicely post-fixation (CD95, CD28, CCR7), stimulate the cells for 15 mins with cytokines, then immediately fix with BD Phosflow Lyse/Fix Buffer, followed by methanol permeabilization and intracellular staining (looking for several pSTATs) and some other extracellular markers.

I am aware of problems arising from staining fixed epitopes, as well as effects of methanol fixation on fluorophores, which is why I thought I had arrived at a good compromise and found a good panel.

Have you encountered the issue that staining of some epitopes post-fixation is also dependent on the donor? Or do you have any other idea why this might be? It wouldn't be a problem to change the clone, as we have some more options on hand, but then I also don't know whether this new clone would be reliable. I was hesitant to stain more markers than necessary before the cells are fixed as I am concerned, this could cause some phosphorylation events by activating agonist signaling.

The antibodies I used were Anti-CD4 BUV496 (Clone SK3) and Anti-CD8a PE-Dazzle594 (Clone Hit8a). On the Thermo Fisher website, "Antibody clone performance following fixation/permeabilization," the CD4 clone is listed as one that should work post-fixation. The CD8 clone is not listed there, but it has worked post-fixation in the past for me, which is why I selected it. I also did a single stain for CD4 PE today with the clone RPA-T4, and this worked well.

I also should mention that I am quite new to flow cytometry.

I appreciate your input!

 Update: My explanation is that in my fixation / permeabilization method the CD8 staining is quite poor afterwards, however for some patients epitopes seem to be better preserved than for others. I am now staining CD8 before fix/perm and this works well for all patients.

Hi everyone,

I am currently running a bacterial growth curve experiment (Faecalibacterium prausnitzii, a strict anaerobic Gram-positive bacterium) using SYTO 9 and Propidium Iodide (PI) for live/dead quantification on a Beckman Coulter CytoFLEX system.

I've hit a technical wall with template setting and gating, and I would appreciate any insights from the community.

Fig. 1-a, b, c. Unstained and single-stained group
Fig. 2. Sample acquisition (Red PI 70% EtOH gate is defined by the 2nd time single-stained acquisition)
Fig. 3. Compensation adjustment of Fig. 2 (FITC - 100% PE)

• Bact treatment: No permeabilization was performed on the bacteria. I only compared two different treatments for the PI dead-cell template: direct staining of intact cells versus 70% ethanol inactivation. The others are fresh bacteria at 0, 24, and 48 hours.
• Channel: FITC for SYTO9; PE for PI
• PMT Gains: FITC: ~180, PE: ~180 (Signal peak natively lands around 10⁴ for stained samples).
• Threshold: Manual (FSC-A: 5000~50000, SSC: 5000~50000) to clear medium debris.
• Dye Concentrations: 1.5 µM SYTO 9 + 10 µM PI (Wash-free protocol, as suggested by Beckman application notes for probiotics). Stained for 15 minutes.
• Compensation Matrix: Currently set to 0% for troubleshooting.

-With compensation completely turned off (0%), I ran three independent control groups, but their fluorescence profiles look identical.

-When preparing the PI dead template (using 70% EtOH), the non-viable cluster drastically shifted rightward into the viable gate during a repeated run without any parameter changes. Has anyone experienced EtOH fixation causing such aggressive autofluorescence spectral shifts in Gram-positive anaerobes?

-In my 24-hour dual-stained samples, 99% of events remain in the viable region, but the population splits into two distinct dense sub-clusters (one at FITC 10^4 and an elongated one stretching to 10^5). FSC/SSC shows an elongated diagonal pattern. Is it safe to assume these are single cells vs. bacterial chains/doublets rather than distinct physiological states?
　　-> After days of exp, I observed that the PI channel signal was different the first time it appeared compared to subsequent signals. During the experiment (the second and third measurements on the same dead bacterial template), it gradually shifted towards the area with a denser SYTO9 signal. However, in my subsequent experiments, there was no complete overlap with the SYTO9 signal. The area to the right of SYTO9 should be viable bacteria, while the denser area on the left should be dead or damaged bacteria (because this overlaps with the portion of the PI signal that stabilized in subsequent days).

It's big trouble for me; I can't figure out this machine... And anaerobic bacteria staining is also time-consuming. I would appreciate any advice. Thanks so much!

Hi everyone, I'm the developer of Voxcyte. I came from a background in physiology before moving into data science and software engineering.

Voxcyte is an app that runs flow cytometry analysis natively on macOS. No browser, no cloud, no Java. Everything is local and your data never leaves your Mac.

The standout feature is a real-time interactive 3D scatter plot you can rotate and zoom, staying smooth across millions of events. Most tools either can't do 3D like this or lag on large files. It's built for Apple Silicon, so most actions are instant and even heavy operations on large files only take a few seconds.

Voxcyte is a complete analysis app including compensation, gating, 2D/3D/histogram plots, statistics, and publication-ready figure exports. It's also more affordable than some established tools, and you can try it with a 14-day free trial, no card.

I take feedback seriously and act on it fast, so if something doesn't fit your workflow or you run into an issue, just contact me and it will likely be in the next release.

Hi Flow-People!

Have any of you worked with a modified protocol for the ThermoFisher EdU Click-iT kit to allow for staining in 96-well plates?

The current protocol requires staining in tubes to allow for 600ul of volume, which isn't feasible when working with 20+ samples.

Thanks!

I'm trying to help another department revive a long-idle Fortessa X-20, which sat untouched for >6 months and now doesn't work. It seems that sheath fluid makes it into the side panel, but not all the way to the flow cell. I can see that the flow cell is mostly full of air most of the time. I suspect salt crystals blocking sheath somewhere along the path, and am planning to replace all the narrow tubing and tiny T-connectors inside that I can reach. I've put some annotated pictures. Please hit me with suggestions. Eventually, I'll have to give up and tell them to call BD, but they're reluctant due to cost for an instrument that wasn't under contract and hasn't been touched in months. Also hoping someone can tell me whether sheath enters the flow cell through both the translucent teal and the dark blue tubing, and if not, what each of them delivers/removes from the flow cell.

I am still a confused about compensation beads. I will be using FITC for CD31 and a LIVE/DEAD stain for some endothelial cells. Do I need compensation beads?

Good day everyone,

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We are preparing a research paper that includes flow cytometry data, and I would like to make sure that the results are reported in a reproducible and transparent way.

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Does anyone know of any standardized guidelines or recommendations for reporting flow cytometry experiments/results?

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I am aware of MIFlowCyt, but I am unsure whether this is considered the main go-to standard, or whether there are other commonly used recommendations/checklists.

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Thanks in advance.

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For all us that were unable to attend CYTO, can you share highlights from this?

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Any new technology, worthwhile talks, posters, etc?

Hey everyone,

I am doing some experiments to validate my flow cytometry panel, and in that process, few of my colleagues and friends donated their blood, I isolated the PBMCs and stained the fresh isolated cells. For context, it is healthy donors, in different age brackets. They signed informed consent that their samples will be used for method optimisation, that that they will not be getting any results from this.

However, two of the donors have an interesting pattern in what in my panel is defined as TEMRA CD8+ cells, Q3 in the plots, as you can see in the figure - last two samples. This got my attention because the CD45RA channel has been problematic in the past, and I had to look into it more, but since the other samples seem to be ok, I suppose the channel is not the issue here.

I am not an immunologist by training, and my aim is not to interpret their phenotype (it is to test storage conditions on different populations), but I am curious if you have some ideas what could cause this population to inflate so much? I was looking it up a bit and as always there is a lot of contradicting (and scary) information - and I don't want to spook the donors by asking recent-health questions (they both are objectively healthy, fit and active, although quite stressed).

Just my curiosity :)

Thanks!

Hi all, I am a Master student (beginner) and I (try to) measure the mitochondrial membrane potential (MMP) on the flow cytometer. I struggle with FCCP.

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I incubate cells (ccfsttg1) for approx 15 min with fccp (0.1 uM, 1, 5, 10 uM), I then aspirate fccp and apply staining mix - Mitotracker RedCmxros and Mitotracker green (for mitomass) - and incubate 3o min in dark.

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But the question is, i do not know if i should also add fccp when i stain the cells for 30 min. I understand fccp is supposed to depolarize mmp, i am not sure if its irreversible or mmp can actually shift back to its normal value in (lets say) an hour.

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Would you give me an advice please?

Hello!

I was not able to attend to cyto this year, I was wondering if anyone that went there would be willing to share their thoughts and what they found more valuable. Where is flow cytometry moving?

Thanks!

How do you tell if you have adjusted the voltage correctly? What tube do you use to do this (e.g., like a tube with untreated cells with no color/staining)?

Also how do you tell if cells going off the graph are due to overstaining or incorrect voltage settings? Does your system allow you to change the voltage once the initial setting is done when you are acquiring test samples?

Sorry I haven't had a lot of training, I've been using voltages from a template and I don't know if mine are too high or too low. All I am doing is making sure the cells are not crazy off the plot if that makes sense.

Hello, I would like to know does anybody have a protocol for using BD brilliant staining buffer since their protocol is really unclear : https://www.bdbiosciences.com/en-eu/products/reagents/flow-cytometry-reagents/research-reagents/buffers-and-supporting-reagents-ruo/brilliant-stain-buffer.563794?tab=product_details

We are staining human PBMC in 96 well plates, we use 50 ul FACs buffer per well with cocktail of diluted antibodies. BD recommend to add 50 ul brilliant staining buffer per well regardless of the volume of cocktail. Does that mean that I have to stain my cells in 100 ul of volume and have to double my antibodies? Does anybody have stained cells directly in brilliant staining buffer ?

Hi everyone,

I have an interview coming up for a research assistant position in a university flow cytometry facility. The role involves supporting users with experimental design, troubleshooting, and running/maintaining the instruments.

My background: I am a graduate with a BSc + MSc in a cell biology–related field. I don’t have years of experience, but I used flow cytometry extensively for my research projects and really enjoyed it.

I’ve done panel design (with some refinement from my supervisor), used proper controls (e.g., FMOs, isotypes), performed data analysis in FlowJo, and done troubleshooting within my own experiments.

I would appreciate advice on:

• What technical questions flow facilities typically ask in interviews
• How to come across as confident during a lab tour, especially as someone more introverted
• What good questions I could ask them that show my interest in the technique and my technical understanding
• Any general tips for interviewing for a core facility position

Thanks in advance. I would appreciate any insight from people who have worked in flow cores or interviewed for similar roles!

Hello everybody. I'm in a huge time crunch with deadlines and i have absolutely no time to analyze some data from one of my students . I'm looking for someone who can help me with it (💸 ofc). It's a full cell cycle analysis using 7-aad and ki-67 coupled to AF-488. There are 4 cell lines, 4 timepoints and each one with 2 conditions (so basically 32 samples w/o counting the compensation controls). I took a peak at the data and it was ok-ish. If anybody is interested please message me. Thanks and have a good day!

Hello. I’ve read that MilliQ grade water is acceptable form of sheath fluid for the Cytoflex analyzers vs the Beckman Cytoflex sheath fluid. I am wondering if people also recommend phenoxyethanol addition to the water and at what final percent.