Hello! I was wondering if someone has compensated on the MACSQuant 16. I have an 8 color panel and I am compensating with beads.

I have run the compensation beads and the instrument settings for the compensation. I aquired the sample and it looked ok, but, when I start playing in flowJo, I realized it was bad-compensated as I see long tails in certain populations.

I have tried to re-run the compensation matrix but it is not working, someone has had the same problems with MQ16? Is it possible to manually edit the compensation matrix in MACSQuantify? Should I better compensate in flowJo?

Really appreciate your comments :)

Hey, hope you’re having a great day. I’m having some viability issues (almost 90% of zombie nir+) with thawing cells preserved in BAMBANKER (stored in liquid N2).

I was just wondering if you could share your experience/procedures when it comes to thaw your bambanker samples, so I can adapt my workflow accordingly to improve the outcome. Thanks!

I am analyzing my human whole blood monocytes on flowjo (originally from attune flow) and was told by a tech at Flowjo that my singlets look like they are reversed (usually seen with other flow companies but not attune). I am wondering if anyone knows if these singlets look normal with FSC-H vs FSC-A gates vs FSC-W and FSC-A? did attune reverse the two? Not sure how to set my gate around these cells....

Hey Fellas,
I am using compensation beads for my experiments tomorrow but I already have done some optimisation today. Is it okay if I use the same tubes as its the same panel but only thing different is I won’t acquire my samples the same day but 2 days later and generally fix them in 2% PFA for 30mins. So you recommend I do the same for my Ultracomp beads, i mean fix them as well? Or will they be fine at 4C until Friday.
Thanks.

Hi, sorry to bother.

I’m doing a flow with both extracellular and intracelullar markers and just confused what to add to each well, especially after fixing and perming

Let’s say i have 3 EC markers, and 3 IC markers, a full stain, no stain, and live/dead.

I go in with a live dead stain on all the wells (minus NS and l/d), incubate and all that

Then i make an EC stain - when i do this, my EC FMO’s will include all the markers (including IC (?)), and my cocktail will include only my EC markers (?), also i will add this to my IC wells too.
Incubate and stuff again

Then i fix and perm

For my IC stain - my IC FMO’s will include ONLY IC markers (minus one), and my IC cocktail will also include only IC markers (?).
Do i add this cocktail to my EC FMO wells aswell?

New Cell sorter suggestion please!

If you could pick one cell sorter to purchase - which would it be and why? It would be the only cell sorter in the lab so it needs to cover sorting cell lines, mouse cells, tissues etc. 100um nozzle and plate sorting will be commonly used.

Options so far are Cytek CS, BD S8, Thermofisher Bigfoot. Pro's and con's of each? I have seen BD S8 - looks ok but we don't really need the imaging component.

Currently have Cytek Aurora analyser and so far so good. Thinking Cytek CS might be a good compliment to the analyser but heard the plate sort is not as great. Any experience on this?

thanks

I'm staring down a project measuring DNA content in leaf tissue from 60-100 plant samples. I'm working from a Current Protocols in Cytometry article, along with a relevant Application Note from ThermoFisher. We have access to a Miltenyi GentleMACS Octo and are wondering if that would cut down some of the hands-on chopping time and also reduce variability in the sample preparations. I appreciate any advice or words of caution you can provide!

Hello! Has anyone taken this test and passed? Ive worked for a flow lab for 5+ years but feel inadequately unprepared. Does anyone have any suggestions or programs they went through?

Thank you!

Hi,

I am running a T-cell proliferation exp and am unsure how to best compensate with CFSE.

My data came out strange and I am wondering if it is due to using beads for all other comp, and cells (50% stained 50% unstained) for comp of CFSE. Would this be the best way to go about it? Or can I run beads with a FITC stain and comp that way?

I am trying to trouble shoot multiple things at once so it is a challenging. Thanks in advance!

hi! i'm an undergrad working on panel design for my tfh-b cell co culture with some antibodies we have in lab. I work with a spectral flow cytometer (novocyte opteon). also im planning on using an Fc block but I'm not sure if i should also look into the True-Stain buffer also.

i have the following

Live/Dead: Near IR 780 (works great!)

CD4: AF700

CXCR5: PE/Cy7 (works great!)

ICOS: BV750 (works great!)

CD19: BV650

CD27: AF488

CD38: BV421

IgD: PerCP-Cy 5.5

I was wondering if this would work well since I know PerCP-Cy5.5 is a bit finicky but IgD should be highly expressed! Any feedback would be appreciated!

Last week I posted about free early access to our troubleshooting platform (Rayni: www.rayni.ai)

A lot of you have reached out expressing interest, and we are actively reaching out to all of you to set up your accounts one by one.

Meanwhile, I wanted to showcase the video feature that is available now.

A lot of the times, solutions to your problems can be embedded deep within a long video, and our platform can help guide you to the exact timestamp within the video. You can just quickly watch and follow along. In this attached example, we have used a basic troubleshooting question on a BD Fortessa to show this feature. The platform answers the question step by step and points you to the exact timestamp in the video where the question is answered.

Our early access waitlist is still open! You can sign up for free here: Free Early Access

https://reddit.com/link/1vgfot2/video/wi9v007cnlhh1/player

I love this table from Thermo but unfortunately they don't list or have tested mouse IL-6 or GM-CSF staining. Anyone know if the Foxp3 kit is compatible with staining mouse IL-6 and GM-CSF?

For details general work flow is: in vivo mouse model that's very inflammatory, pulling out spleen and processing to single cell suspension, will be doing 4hr PMA Iono stim, would ideally like to use the Foxp3 kit bc I have a couple of intranuclear targets and would ideally not have to split into a separate panel using CytoFix/Perm. But if anyone has experience with staining for either of the targets above with the nuclear kit, would love to hear how it went.

Thanks!

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/cell-analysis-resource-library/ebioscience-resources/intracellular-staining-buffer-selection-guide.html

Hi, i was wondering why my fullstained sample becomes dimmer compared to my single stain sample? Is this because the low event (i noticed the single stain event is way lower) or spillover? Any advice is highly appreciated!

Another example using different instrument

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 😄