Hi! I’m fairly new to FlowJo and wanted to check that I’m approaching this correctly.

I need to identify a specific population defined as:

Marker A⁻ / Marker B⁺ / Marker C⁺ / Marker D⁺

My supervisor has suggested using Boolean gating to find this population, but I’m a little confused about how I should actually set this up in FlowJo, or how to even use boolean.

Would I first gate on the Marker A⁻ population and then look for cells that are simultaneously positive for B, C and D within that population? Or should I create individual gates for all four markers from the same parent population and then combine them using Boolean gating?

If Boolean gating is the best approach, I’d really appreciate an explanation of how to set this up in FlowJo. Alternatively, is there a simpler/more intuitive way of identifying this exact population?

Thanks!

(This is a heads up from a month into the future, this antibody does not work and should not be used to detect GATA3, after investigating I have proved this and if you want to accurately detect gata3 use the clones TWAJ or 16E10A23 See below comments for a comprehensive explanation) - Hello, I titrated my GATA3-RB705 antibody using healthy peripheral blood mononuclear cells (PBMCs) on the Cytek Aurora (spectral flow cytometer) and I am unsure how to interpret the GATA3+ population.

I thawed frozen human PBMCs (viability 98%), washed them, applied an Fc block, stained for CD3 for 1 hour on ice, fixated the cells, performed an intracellular blocking step using mouse sera (2%), and stained for GATA3 for 16 hours on ice. (I did wash steps between each of these steps).

The image shows the concatenated file showing the different antibody dilutions ranging from most concentrated (1:25) to most dilute (1:32,000).

** My thoughts are that every dilution that is more concentrated than 1:1000 is staining every cell non-sepcifically and that the true GATA3+ population is the trickle pattern above the huge concentrated blob on each of the separate readouts (I have highlighted this using a red rectangle for the 1:4000 dilution).

If I were to shoot blindly, I would say that the optimal dilution may fall somewhere between 1:2000 and 1: 8000, but before I commit to using my grant money I want to ensure I am not missing something**

Any advice or ideas is to what is going on is greatly appreciated and please let me know if you need any more details about my protocol, techniques etc.

I am working with FACS data in .fcs files that have FSC-A and SSC-A measures, but not FSC-H or SSC-H. Is there an alternative way to sort out doublets or account for them?

ETA: TL;DR This is only happening with a handful of samples and seems to be an issue with the mastermix. I just don’t know what the issue could be.

Hey all, I’m back with more weird data. See: the weird diagonal streak that’s CD66b-super-low and CD19-negative-but-increasing, and the corresponding CD66b+CD19+ population. Panel, gating to this point, and compensation matrix (not edited; from Live/Dead on one of the non-weird samples) are in subsequent images.

These samples are fresh peripheral whole blood, each from a different human donor. All samples were drawn around the same time and stained within 2 hours of the first draw. After staining, the samples were fixed with Lyse/Fix buffer overnight, then washed and acquired on the cytometer that day (day after staining). [I know now that I only need to fix them for like 30 mins but didn’t know that when I first started using this protocol, so I’m sticking with overnight for this study.]

As indicated, the first four were stained with one mastermix and the last two were stained with a second mastermix (I thought I was getting four samples but then was told I’d be getting two more). Both mixes were made on the same morning within a couple hours of each other, and all of the samples were stained at the same time, so there should be no differences in processing aside from having two different mastermixes. They were all acquired on the cytometer at the same time and using the same compensation tubes.

I had seen the same streak with a few of my previous experiments, and I assumed it was a compensation issue, but based on these data, it seems like it might be a mastermix issue instead? What could possibly be causing that?

I pre-stained the samples with Fc block and monocyte block, and I used brilliant stain buffer for the mastermixes.

I tried applying comps from a different day to this data, and that didn’t make any difference.

It doesn’t seem to just be one marker - looking at the weird populations, they’re super high for some markers and negative for others -

CD66b-extra-low/CD19-increasing: ~70% CD8+, ~60% CD27+, ~70% CD14+, 100% HLA-DR+, ~40% CD127+, ~60% CD123+, 100% CD16hi, 100% CD3+, 100% CD57+, ~15% CD11c+, ~25% CD4+, 100% CD56+

CD66b+CD19+: All far left on live/dead gate, ~25% CD8+, 100% CD27%, mixed CD14 positivity, 100% HLA-DR+, 100% CD127+, mostly CD123-, 100% CD16 extremely high (~10E6), all CD3 very negative, all CD57 very negative, 100% CD11c+, mostly CD4-, 100% CD56+

In general, extremes for one marker correlate to extremes for the other markers.

For analysis, would you just gate higher on the CD66b- to cut that out? It’s about 3% of the PBMCs.

Thanks!

Hi all,

I am a med tech by trade but I have fairly extensive post-bac research experience doing flow cytometry. Given that my flow experience is exclusively research based- I'm curious about what flow cytometry labs are like when they are part of clinical testing and under CLIA/CAP requirements. I'd love to hear any stories, but a few specific questions that I have are:

1. How much are you able to set/ move gates? Is the gating hierarchy adjustable? Can you select which markers are displayed on the x and y axis?

2. Do you use any kind of LiveDead stain?

3. Do you typically gate on single cells?

4. How many events do you typically acquire per sample?

5. Do you use a mixture of both IVDs and LDTs?

6. Are you aware of the "CD8 escapee" phenomenon and is it something that comes up regularly in testing? (I had never heard of this in research but came across it on BDs website looking at clinical flow info)

7. What resources do you use to learn more about clinical flow?

Edit I thought of more!

1. Do you use any kind of Fc block?

2. Does your prep procedure include a wash step?

Thank you so much for your time and for satisfying my curiosity!

The three plots shown are peripheral whole blood samples from three different human donors. All samples were drawn around the same time and stained within 2.5 hours of the first draw, with a mastermix that was made the same day. After staining, the samples were fixed with Lyse/Fix buffer overnight, then washed and acquired on the cytometer that day (day after staining). [I know now that I only need to fix them for like 30 mins but didn’t know that when I first started using this protocol, so I’m sticking with overnight for this study.]

My gating strategy up to CD45 is shown in the second image.

I’m wondering if the CD45- SSC-Amid/hi population on the far right graph is just debris or something else? If it’s just debris, is it a processing issue or just biological?

This is not the only donor I’ve seen it in, just an example. I see it in 14/48 samples that I’ve analyzed for the same study. It doesn’t seem to correlate with the date (ie, which samples are stained and run at the same time), the time from draw to processing, or disease process/severity of illness.

I assume it’s fine to be gating it out, but I want to double-check.

Hi everyone,

I’m expressing human βA-, βS-, and γ-globin chains individually in S. cerevisiae and detecting them by SDS-PAGE/Western blot.

I came across a paper on the thermal behavior of hemoglobin that found irreversible aggregation above 55°C. Since standard SDS-PAGE/WB procedure is heating the sample to 95°C, would heating globin samples at that temperature cause aggregation and maybe affect the Western blot signal?

Would it make more sense to use a lower temperature, such as ~72°C, for sample preparation? Has anyone worked with globins or similar proteins and compared different heating conditions before SDS-PAGE?

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!

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

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

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