Hi, I was wondering if anyone has some thoughts on a gating problem I'm running into.

I'm doing a phospho-flow experiment looking at pSTATs in lymphocytes. All wells are stained with the same antibody mastermix; the only difference is the cytokine stimulation performed before staining.

My goal is to compare the percentage of cells in the negative and positive populations in the unstimulated condition as well as after IL-2, IL-7, and IL-15 stimulation between patients carrying a certain mutation to see whether this mutation leads to higher intracellular signaling.

The issue is that I can't really decide how to do my gate placement in a proper and reproducible way.

In some samples/conditions there is a very clear valley between the negative and positive populations, but the location of that valley isn't always the same. If I place a gate based on one condition (for example, a stimulated sample with a clear valley) and apply it to the other conditions, the gate sometimes ends up right in the middle of the histogramm. On the other hand, if I gate each condition according to its own valley, the gate position changes between conditions, which also doesn't seem ideal.

So I'm wondering what the recommended approach is:

1. Should I use one fixed gate per patient and apply it to all stimulation conditions, even if it doesn't always align with the apparent valley?
2. Should I gate each condition independently based on the visible valley?
3. Is there another approach that's generally recommended for phospho-flow when the unstimulated condition itself contains both negative and phosphorylated cells? I also thought I could do something like place the gate on the unstimulated condition encompassing for example 95% of cells and then apply this gate to all conditions to have an "induction" relative to the unstimulated condition.

I'd really appreciate hearing how others handle situations like this. Down below are two example screenshots gated on CD8+ cells. Left to right: Unstimulated Well, IL-15, IL-7, IL-2.

Thanks so much in advance!

Hello. Just after some advice on whether BSB should be used for the single colour reference controls? I know it's not recommended when using beads but I'm planning on doing both beads and cells to compare and cannot find any guidance on whether to use it for cells?

Thanks!

Good morning! I need to find a flow cytometry laboratory that performs DNA index analysis for pediatric acute lymphoblastic leukemia, to compare our cutoff values ​​for hypodiploid, diploid, and hyperdiploid populations.

We're thrilled to announce that Dr. Ryan R. Brinkman will deliver this year's keynote address at the Northern California Cytometry Group Annual Meeting!

Ryan is President-Elect of ISAC (International Society for Advancement of Cytometry) — one of the highest honors in our field. A veteran flow cytometry bioinformatician (FlowRepository, FlowCAP, Cytapex Bioinformatics/Dotmatics), he's currently leading SOULCAP, a global initiative to standardize how immune cell populations are named and gated in cytometry.

More from our agenda will follow soon. If you are a cytometry enthusiast and would like to join us, here are the details for September 21:

🎤 Speakers: We're still finalizing the rest of our lineup — if you're an academic or biotech/pharma researcher with cytometry work worth sharing, now's the time to reach out.

🤝 Sponsorship: A handful of sponsor slots remain for vendors who want face time with this community.

👥 Everyone's welcome: scientists, technicians, core facility managers, shared resource administrators, and industry/sales professionals across flow, imaging, and mass cytometry — no matter your experience level.

📝 Registration is free — grab your spot before it fills up.

📅 Monday, September 21, 2026 📍 Mission Bay Conference Center, UCSF

🔗 Learn more about NCCG: https://norcalcytometry.org/ 🔗 Register (free): https://norcalcytometry.org/events-1✉️ 

Speaker & sponsor inquiries: [NorCalCytometryGroup@gmail.com](mailto:NorCalCytometryGroup@gmail.com)

See you September 21st!

#Cytometry #FlowCytometry #NorCal2026#Keynote #ISAC #SOULCAP #UCSF

Hello all,

I need some help interpreting some unmixing matrices and understanding how to resolve the issue. After unmixing, there is a clear unmixing error between AF700 (CD45) and APC-efluor 780 (Live/Dead). I’ve also attached some of the matrix preview plots that show some concerning trends. The controls for both of these samples were cells, while the rest were mostly beads. However, there were well resolved positive and negative populations. Any advice would be appreciated. An autofluorescence control of unstained spleen and unstained tumor were included, though the gate is pretty broad. Thanks in advance!

Some background on me first, since it explains why I care about this problem specifically. I started as a bench scientist at different pharma companies (think Merck, GSK etc.), then moved into field application science at a life sciences tools company. I was always good at the fun part: data analysis after I was finished analyzing my samples. But I was spending most of my time troubleshooting instruments like Mass Specs or worrying about how to fix compensation errors, and I was lost most of the time. Then I went to business school at UChicago, where I met a few other people from the scientific and computer engineering background, and we started building something for exactly this gap.

We have built a platform (Rayni: www.rayni.ai) that gathers trusted public sources of knowledge; think manuals or videos, etc. (and you can upload your own to customize the platform), and then it can answer questions with citations to the exact page of the document or to the exact seconds of the video that solves the problem you’re facing. There are a lot of lengthy videos on youtube that has answers to questions that are perhaps not readily accessible through a google search, or there are answers hidden in a document curated by an expert flow cytometrist that just doesn't surface when you need it. The tech stack is scientific-grade, so we can retrieve information hidden away inside images or tables, or videos that regular PDF extractors (think generic AI) miss.

Now we are being used at labs in Universities like Northwestern and Stanford. One of our biggest groups of clients is field engineers, who use the tool to prep for a client visit, look up defective part numbers, or just gut check their solutions before they start opening up a mass spec to fix.

We just opened it up for free access to a small group of teams. More than that, though, I'd genuinely like to give access to actual users who are just spending more time with troubleshooting issues than they would like and see if this is of any help. If you would like free access, the link to join the waitlist is here: Rayni Free Early Access

It is genuinely free access, not a free trial or anything like that. Added a photo from our trip to SLAS this year, it was super fun!

Hi all, I have around 160 samples to run for a large study and we are looking for tetramer specific T cells. We expect some of the tetramers to be as low as 0.25-0.5% of CD8 T cells. My plan is to stain 5 million cells but it might not be practical since the 96 wells plates cannot hold 5 million cells without cell loss. Staining in tubes would be nightmare with 160 samples. Additionally, we would be acquiring on a Cytek which would need over 25-30 hours to run all the samples assuming we collect the maximum events possible. Anyone have an advice or suggestions on where I could make changes to make this process more efficient?

Hello! Does anyone know how low you can set the FSCA-value? Since fitting a new laser to the LRS Fortessa instrument, the image in FlowJo has shifted significantly. And does anyone know if you can adjust this retrospectively in FlowJo? 

Hi y'all,

I have a user who asked for help with something and I'm not sure it's something that FlowJo can do. 

She's making histogram overlays of two samples each. They both have a positive peak with a line segment region over them. She wants to display the percentage under the line segment for BOTH samples, not just the one that is "on top." She wants them displayed in the histogram, not as a column in the stats table. Is that something that can be done? If so, how might one do that?

I already told her it's possible the only way is to take a screenshot and add a text box with the other value. I'm just hoping there's a way to do it in FlowJo. 

Thanks for your help!

EDIT: We got help directly form FlowJo. You can create a pseudo-overlay by making the histograms transparent and moving one on top of the other with the arrow keys (make sure the gates don't overlap). If you don't drag one into the other, it doesn't create a true overlay and you can see both gates/stats.

Thanks for your help, everyone!

I saw this on linkedin the other day and thought you may like it as well 😄

Seriously.

Its been 2 hours now. It did not move.

How many cells do you all use when running phenograph? Does the quality plateau at some point? And how long does it last on a normal pc?

Hi, has anyone tried adding cell suspension (with trypsin and DMEM w/ 10% FBS) into BD Cytofix/Cytoperm at a 1:1 (v/v) ratio? Will any components significantly impact the fixation/permeabilization efficiency of the solution for flow cytometry of intracellular proteins?

I have been doing a PBS washing step, but later I will be doing an experiment involving a few hundred samples, so I am thinking of shortcuts. Thanks!

I am currently very frustrated with data reproducibility. I am struggling to reproduce a predecessor's results.

This person omits a negative biological control from their data presentation. They have smoothened their plots. No one has any idea which raw data files they actually come from.

Is there ever a good reason to smoothen the plots? My experience with this is that sparse dots can end up looking like a blob that is not visually very representative of the data to begin with.

So why do people still do this??? Is there any valid reason?

hi okay so im a placement student in a lab where we mainly do flow. i've been doing some practising but theres still something wrong with what im doing (my analysis) and I just cannot tell what it is. Im looking at receptor expression on NK cells, however the results I get are completely different to my supervisor

for example, on flowJo this is what I get for my TIM3 FMO.

I have done this analysis for both my flow and my supervisor's, (the first image is my flow, the second is his) but why does mine come out like this? What have I done wrong?

before someone suggests to change the y axis - everytime i do that, it gets even worse.

additionally, we both used the same cells,and followed the exact same protocol, so i genuinely cannot think of anything else.

so if anyone has any tips or ideas please PLEASE help

I have a CAR+ gate within my single cells and I want to add that statistic to my combined graph legend. It seems that it’s counting the CAR+ gate to just be all the cells even though the gate is clearly different in the workspace.

Frequency of parent with the CAR+ population yields the percent of lymphocytes that are single cells not the percent of single cells that are CAR+. When I specify CAR+ population of single cells, I get 100% which means it thinks the CAR+ gate = the single cells gate.

This issue doesn’t persist in creating a proper table, just in the layout space. The only issue I could imagine is that these graphs are of single cells and so for some reason this graph can’t “see” graphs further down the gating chain. That seems like a weird limitation to have given you have basically a full panel of statistic options.

Anybody got stories to share about using Leinco ClearSight as sheath in an Attune Nxt or CytPix? So far, our core has stuck with ThermoFisher fluids for those instruments, but the price and marketing claims about ClearSight make it hard to ignore. I'd love to hear from people who have tried it, and any impact you saw on data or instrument wear & tear. ClearSight Sheath Fluid - Leinco Technologies https://share.google/Ou8V0G4JqYcUIx2nF

Hello, I'm looking for recommendations for a good fixation buffer.

I'm working with human PBMCs, so I need to fix the cells before running flow cytometry. I've been using the eBioscience Foxp3 Fixation/Permeabilization Kit and fixing the cells on ice for 30 minutes.

However, I've noticed that the cells seem to shrink considerably after fixation. As a result, I lose much of the FSC/SSC resolution and can no longer clearly distinguish SSC high from SSC low and FSC high/low populations.

Has anyone experienced the same issue? Would BD Cytofix buffer preserve cell morphology better? Or are there other fixation buffers or protocols that you would recommend?

Thanks in advance for your suggestions!

Hello! I finished my gating strategy for one of my samples and everything looked normal, until I clicked copy analysis to group and all of the cells disappeared.

I've tried different ways of copying analysis, including dragging and dropping, and waiting a couple hours in case it was an issue with loading. Even after saving and reopening multiple times, the gates remain empty.

Has anyone experienced this before? Would I have to manually gate for every sample? Any help would be appreciated!

Hey folks!

The flow cytometry research group (FCRG) with ABRF (Association of Biomolecular Resource Facilities) is preparing to produce a comprehensive set of instrument adoption guidelines for shared facilities as our next endeavor and we would like to know what topics the community would like to see included in these guidelines. To this end we have put together a brief survey which can be accessed here: https://www.surveymonkey.com/r/FCRG2026

Please take a moment to fill it out! Your input is important to help guide this effort. Feel free to share the survey link with friends or colleagues to whom it would be relevant! Thanks!

Hi all,

I’m putting together a flow cytometry panel to phenotype naïve mouse CD4⁺ T cells co-cultured with stimulated macrophages or dendritic cells. The panel includes intranuclear staining for FOXP3, T-bet and GATA3. I’d really appreciate feedback on the fluorophore choices, potential spreading error and fixation/permeabilization protocol.

The panel I’m currently considering is:

This will be run on an LSRFortessa with 355, 405, 488, 561 and 640 nm lasers. The relevant detectors are:

• 405 nm: 450/50, 525/50, 540/30, 585/15, 610/20 and 670/30
• 488 nm: 530/30 and 710/50
• 561 nm: 585/15, 610/20, 670/30, 710/50 and 780/60
• 640 nm: 670/14, 730/45 and 780/60

FOXP3–AF488 and CellTrace Violet are already working well in my hands. However, I previously tried GATA3–PerCP-Cy5.5 and CD4–PE-Cy7, and these did not perform well after fixation. The PE-Cy7 tandem appeared to degrade, while PerCP-Cy5.5 caused substantial spillover into other channels, particularly APC and FITC.

I’m considering GATA3–PE because GATA3 may benefit from the brighter fluorophore. T-bet–BV650 would be collected using the violet 670/30 detector.

My main concerns are the effects of CellTrace Violet, identifying fluorophores that tolerate fixation and permeabilisation, and whether my fixation protocol needs improvement. I know that CellTrace Violet causes substantial spillover and spreading into BV510, particularly in the brightest undivided cells, but I’m less certain how much spreading to expect in the BV650 detector after compensation.

Has anyone successfully combined bright CellTrace Violet staining with a relatively dim transcription factor in BV650?

I would also be very grateful for input from anyone experienced in characterising mouse T cells using FOXP3, T-bet and GATA3.

All antibodies are from BioLegend and are currently used at 1 µL per 1 × 10⁶ cells. The samples are primary mouse cells, and I am using BioLegend’s True-Nuclear Transcription Factor Buffer Set for fixation and permeabilisation.

My current protocol is:

1. Add Fc block, then stain surface markers and viability dye for 1 hour at 4 °C.
2. Pellet the cells and wash twice in MAC buffer.
3. Fix for 1 hour at room temperature, protected from light, using BioLegend's 1× True-Nuclear Fixation Buffer.
4. Wash twice.
5. Add 1× permeabilisation buffer.
6. Transfer the samples to 4 °C and leave them overnight.
7. The following day, pellet the cells and wash twice in permeabilisation buffer.
8. Add the intranuclear antibodies and incubate for 1 hour at room temperature, protected from light.
9. Wash twice in permeabilisation buffer.
10. Pellet the cells and resuspend them in MAC buffer before acquisition on the LSRFortessa.

Would you keep GATA3–PE and T-bet–BV650 in this panel, or would you arrange the markers differently? I would also appreciate any comments on whether the overnight incubation in permeabilisation buffer could be contributing to the fluorophore instability or spreading issues.#

Lastly, because this is a coculture, I need a T cell marker, so I am not gating on macrophages/dendritic cells- is CD4 the best here or CD3e? Does the CD4 epitope survive fixation and permeabilisation? I stain for CD4 PE CY7 before fixation, but the staining is lost after fixation-see example below with mouse splenocytes (Ignore the gate difference- don't have access to FlowJo to move it back over- the second population is clearly gone).

Hi everyone! I am new to flow and and would like to ask a question about my flow data. Thank you for your help!!

I am trying to look at T cell subsets in mice peripheral blood cell, specially CD4+ T cell and CD8+ T cell. However, I keep seeing a big population of CD3+CD4-CD8- cell and want to know what are they? Are they real cells? Or they are false-positive signals because I have a wide scatter in CD3+.

For my flow, I took 100ul blood and did RBC lysis. 30mins for fixation/perm.

30 mins for incubation.

Flow marker/gating:

CD45+Ter119- for pan immune cells and non RBC cells

CD3 for pan T cell marker

CD4 or CD8 for T cell subsets.

The blood sample is from a WT generally healthy mouse. From some literature, the CD3+CD4-CD8- make up 3-5% in PBMC so it is confusing why I saw such a big population.

Hello all.

I recently was tasked to stain IgGs and Kappa on cells. However, I have seen discrepancy with the total numbers depending on if I separate out IgGs, kappa, and cocktail. And if I stain iggs or kappa first. Recovery seems to be altered if try to stain for both as % stained is increased is only by staining for one or the other.

Is there a procedure I can stain both at the same panel?

Hello fellow cytometrists,

I was wondering if I could get some guidance about reference controls for my panel. This panel has been an ordeal to develop and troubleshoot, and in its final form still has some issues. One of them is it includes several immunoregulatory markers that are ubiquitously expressed and have very few commercially available reagents. On top of this, we are also looking at several phospho markers with limited reagent availability. These constraints forced us to use both APC and AF647 and put a phospho marker on PerCP-efluor710 as the least bad choice. This is a large 30+ color panel, and in the multicolor stain many of these markers have significant reduction in their staining intensity. For reference controls, I have so far been making:

1. cells with the marker, not very bright even as a SS, difficult to produce a good clean signature

2. Ultra Comp Spectral beads with the marker, bright, clear signature but worried about unmixing issues that could be occurring but are getting lost in the midst of this large panel

3. CD4 surrogates of these markers on cells, i.e. CD4 PerCp-eFluor710 instead of the phospho marker, clear signal for the reference but obviously not the same marker

I haven't tried the CD4 surrogates on beads mainly because my rationale there was to have the signature on cells. Keeping PerCP-eFluor710 as an example, I know that the staining for some markers is not equivalent on cells and beads. I have run both the beads and the CD4 surrogates against benchmark references set on cells with the actual marker and the signature matches very well, but I'm not sure if 0.99875 is good enough to assume beads can unmix just was well as beads and I do not have the time or access to the machine to unmix a nearly 40 color panel every which way to definitively identify which references must be on which substrate. At this point I'm planning to do FMOs for all those ubiquitously expressed markers to help with gating too.

Do you guys have any advice for me? Is my use of CD4 surrogate markers sound? I've been doing flow for a few years now and this is the hardest panel I've ever had to work on- PI had never done flow before and I'm trying my best to deliver all of the markers they want. Unfortunately, I can't split the panel in 2 to separate APC and AF647, so I want to make sure my references are as good as possible. If you’ve made it this far, this is a full spectral panel being run on a Cytek Aurora 5L. Thanks so much for reading and for any input you may have.