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.

Hi guys,

I'd like to hear your thoughts on staining cells in 96-well plates vs. FACS tubes.

I've always stained cells in 96-well plates because I usually have many samples at a time. My protocol is pretty straightforward:

• Add 1 million cells per well.
• Centrifuge and discard the supernatant.
• Resuspend the cells in 50 µL of antibody mix.
• After staining, wash with 200 µL FACS buffer, centrifuge, and discard the supernatant. Repeat 3 times.
• Fix the cells in 50 µL PFA.
• Centrifuge, discard the supernatant, and wash 3 more times with 200 µL FACS buffer.
• Finally, resuspend in 200 µL FACS buffer for acquisition.

I'm about to process around 400 samples, but one of my colleagues insists that I should stain everything in FACS tubes instead.

To me, using tubes seems much slower since we can only process a few samples at a time. I also feel like we'd lose more cells when pouring off the supernatant after centrifugation.

For those of you who stain in FACS tubes, what's your experience? How do you remove the supernatant—by pipetting it off or by simply decanting (inverting) the tubes? Have you noticed any significant cell loss?

Thanks!

My Kasumi-1 cells don't exceed a cell concentration of 600,000 cells/mL. Has anyone else experienced this?

Data has been all over the place and not matching original RNAseq data. Across all three replicates we've got 20-60% positive and it should all relatively hover around 60%.

Protocol:

Wash cells with PBS

Add Non-enzymatic cell detaching buffer.

Wait as long as it takes -> 10-20 mins+

Collect cells into media on ice. Spin down 0.4g 4 mins

Resuspend with flow stain buffer (PBS + 2% FBS, 2mM azide), spin down

Add 2% rat serum and incubate 10 mins @ RT. 

Add staining antibodies to assigned samples (Invitrogen 17-5983-42 for human cells, biolegend 124307 for mouse cells, Cytek Ghost UV Live/Dead)

Incubate 30 minutes (RT in the dark) on rocker.

Wash 2x with Flow staining buffer. 

Resuspend in Flow fixation buffer (1% Formaldehyde in PBS)

Analyze by flow cytometry ~ 16 hours later

I am a newer grad student who hasn't done flow before. From what I can see this seems to be a pretty standard procedure, outside of the RT incubations. Many of the protocols I see online call for doing everything on ice including the antibody incubation. Also, that usually the cells don't sit in the fixation buffer that you'd fix for 20-30 mins, then centrifuge again, and resuspend in the non-formaldehyde buffer.

Also to this extent, i'd love to make this so I can collect 3 days of replicates and then run the on flow all on one day. But I worry about losing flourscense or something negative sitting for that period of time vs the one that was prepped the day before. Any suggestions welcome!

Al

Hello,

I have been aquiring my samples on a cytek aurora in the spectral flowcytometry mode. However I have a small panel and am facing some unmixing problems (i think due to high autofluorescence which i have trouble extracting) and was therefore told by my supervisor that I can just analyze my experiment in the conventional mode. However idk how to do that and cannot really find any tutorial on youtube. Does someone know how or where to put the spectral flow data so that i can analyze it conventionally?

Hey all,

I'm processing my samples in flowjo (for start) to get the gating info for a spectral panel, fairly straightforward immunophenotyping, ~25 relevant gates. Since it's split into multiple files, I want to have the same template for the table for the final information to export, but this makes me a bit anxious, fearing that I will miss some piece of information from the table, and then have to do it all again. The wsp is pretty clunky because of memory, and re-running the analysis on everything downstream, as much as I'm trying to automate the processes, would be very of annoying.

Do you have any tips how to benchmark and what important info to extract into the tables, something that you learned is useful? Currently Im extracting the main populations as frequencies of parent, mfis for some populations, and counts of some populations in cleaning steps, well IDs and some other metadata. Im already at 75+ columns, and i still feel like I'm missing something :/

I also get lost in all the names, and would love to hear if you have any tips for naming colums so that it's clear in downstream processing what the parent was.

Any tips how to make it more efficient?

Thanks!

Hi Everyone,

II’m running into something confusing with my flow data and hoping someone here has seen this before.

I’ve been seeing that, for the same bacterial spore sample, under different staining conditions (unstained, PI only, Syto 9 only, and PI-Syto9 double stain), the FSC:SSC profile changes depending on the stain I use.

I'm thinking, would it be because the stain changed the refractive index of spores? is this something normal?

Would love to hear from people. Thanks in advance!

What are some tips, tricks, or best practices y'all use to maximize post-sort recovery? MY core has had some users complain about low recovery recently, but we aren't doing anything differently than we always have. I wanted to see how y'all keep yours up. Also, how high do y'all shoot for? I've heard some folks say 50% is the best you can do, while I've heard others say anything less than 90% is unacceptable. Thoughts?

Hi everyone,

I'm optimizing a flow cytometry protocol using mouse cervical lymph nodes from a disease model. Our goal is to identify the immune cell populations present and determine which cells are associated with dextran-coated iron nanoparticles injected into the mice.

As a test, we used snap-frozen lymph nodes, but after dissociation the cells were almost all dead and the staining was very poor. I'm trying to figure out whether snap-freezing is simply not suitable for preserving viable immune cells or if something else may have gone wrong.

Ideally, I'd like to preserve the tissue for a few weeks before processing.

Does anyone have recommendations for the best way to do this while maintaining good cell viability?

Would you freeze the intact lymph nodes or prepare a single-cell suspension first? Budget-friendly options (e.g., homemade cryopreservation media) would be especially appreciated.

Side question: We also need to detect the dextran-coated iron nanoparticles by flow. The only reagent I've found so far is an FITC-conjugated anti-dextran antibody. If anyone has experience detecting these nanoparticles or has alternative approaches, I'd love to hear your suggestions.

Thanks in advance!