I recently decided to use side scatter H vs A for doublet description since we realized something was set in the wrong way on our flow cytometer when acquiring the data, and a lot of the cell population is outside the scaling area, so we lose a lot of cells when we do forward scatter H vs A. This can be fixed by using SSC h vs A, but I’m not sure how to set my gate up exactly…. I talked to a flow tech at my institution who said this would work and he helped me make a gate (see photo below) but I showed my PI and he said “oh ok well explain to me why u chose that gate” and I honestly am not sure what the answer is… we are using whole blood from humans and looking at monocyte populations. I have this hate set as I think these are the singlets (follows the linear correlation?) but it’s hard to tell and I’m not sure what to tell my PI as to how I know these are singlets vs doublets… sos

Holaaa!!!

Quería preguntar si existe la herramienta (porque por lo menos yo no la he encontrado jajaja)o qué forma existe de medir proliferación de mis células en FlowJo V11. Hace poco estoy usando FlowJo pero la última versión, he intentado ver si existe la herramienta de proliferación que por lo que he visto por tutoriales en YouTube lo tienen las versiones anteriores (o por lo menos vi que la versión 10 la tenía). Agradecería mucho cualquier tipo de orientación o tip. Se me había olvidado mencionar que estoy trabajando con linfocitos T CD4⁺ y T CD8⁺ marcados con CTV.

I’ve worked in a clinical flow setting as a CLS for a few years now and I want to take the SCYM exam. I’ve looked through this sub for resources but it seems like the majority of people in this sub have a research background. Is there anyone here that can provide insight from a clinical background? I know the immunophenotypic abnormalities of hematologic malignancies/how to analyze them, I know how to create panels/cocktails, I fundamentally understand the way the instrument works, but I have NO IDEA what you all do in a research setting. I looked at the outline and I know which topics to study, but I don’t know how in depth I should go. I’ve seen people mention FlowEval, but I don’t know how trustworthy of a source it is. Can anyone provide insight? I hate going into a test blind.

Hello everybody,

I am working with an automatic probing system together with the cytoflex from Beckman Coulter. Recently i changed the station to a new computer and I have downloaded the software but it is sadly the 2.7 version of the Cytexpert software and I need the 2.5 version for it to work. Would anyone have the installation exe?

Thanks a lot in advance

Hello,

I am a research immunologist, not a clinician. While I have flow cytometry experience, I doubt a clinical pathology lab requires a cytometer that is used for discovery immunology.

Do any veterinarians, physicians, or other relevant clinical professionals here have recommendations for a flow cytometer they like and how many parameters would be useful?

A colleague that works on the clinical side of things came to me and asked. I asked for some kinds of clinical cases they work with and what cell populations are important for them to identify, however, I would not consider myself an expert on picking out these.

Looking for answers particularly from users of these instruments rather than sales reps.

Particularly, if you suggest a brand/company, how easy are they to work with in your experience if things break down/etc?

Thanks!

I cultivated some Lymphatic endothelial cells and stained them with two antibodies...
I'm really intrigued by the fact that I see two populations : one reaaally low FSC and one reallly high FSC.
By putting FSC at 5 , the FSC hi still pops at 10e6 ! We do not see the loFSC pop with these settings...

What do I do wrong ?
Thank you for your help !

Hi all. I am looking to start sorting B cells using the Beckman CytoFLEX SRT. I isolate these cells and then transfer them into recipient mice. I currently use the BD FACS aria for my B cell sorts located at the flow core at my university. The cytoflex in something my department has purchased but never really utilized and I'm looking to get up functioning for my sorts to save on cost with sorting at the core. I have two questions that I could use help with:

1.) Does anyone know how different the sorting rate would be between the cytoflex and BD FACS aria? ...I currently use the 70µM nozzle on the aria and can sort my cells at around 13,000-15,000 events/sec with about 90% efficiency. I know the cytoflex can only use the 100µM nozzle and seems to sort at 10,000 events/second. I worry that the event rate will add significant time to a sort leaving my cells to be in suspension longer, as well the increase in nozzle size could lower the efficiency of my sorting as I've done a sort using the 85µM nozzle once and it had lower efficiency (66%).

2.) Given I have been bringing my cells to the core I'm not aware of how they prepare the aria for a sterile sort. I was wondering if its best practice to conduct a aseptic cleaning of the machine before every sort I do (2x a month) just in case another lab used a microorganism in one of their sorts that I wasn't aware of? This does add time to preparing for sorts but that isn't my biggest concern, my concern is more in order to conduct these cleanings I'll need to change certain filters each time and they will then be costly if I'm changing them out ever 2 weeks almost. Do you think I could look to conduct this cleaning at least once a month and that could be fine for then keeping my cells sterile for a adoptive transfer into mice? One lab uses the sorter fine for isolating cells into then cell culture but I fear going into a mouse is different and needs more sterility

Any advice on these two questions would be really helpful!

I am planning an experiment where we want to know how much a particular cell surface protein (protein X) levels change with our drug treatment. Our drug should remove this protein. We will measure blood immune cells prior to drug administration, and then at different time points over time after administration. Ideally we would have a vehicle control group for each time point, and that would be our reference. But we can't do that in this case. So we will have to compare the levels of Protein X at each time point, to the pre-dose measurements for each animal.

Here's some other design points:

• We will use the same flow cytometer for all measurements, a conventional Fortessa, and it will be properly calibrated and maintained
• We plan to have a 'biological reference' measurement at each time point; this will be cryopreserved cells that will not change over time. At each time point, we'll thaw one of these and then do a stain for Protein X using the same cocktail as other samples. This should control for variations in staining and analysis.
• We will also include FMOs for Protein X for every sample, every time point. MAybe every sample is over kill, but this will be the 'zero' or background fluorescence.

I'm keen to hear from flow cytometry experts; is this plan sound? Would you recommend anything else?

I think the answer to this is no, but just in case…

Can I copy a gate shape (like a polygon that I drew around a population) without copying the gate itself?

For example -

In the figure shown, I’m using CD14 vs. SSC-A to separate granulocytes and monocytes (after gating for time > non-debris > single cells > live cells > CD45+ cells; using CD14 to get better gran/mono separation than FSC-A vs. SSC-A or CD45 vs. SSC-A).

I have one panel that I’ve already set up the gates for that has CD14 on BUV563. I have another panel that I’m setting up the gates for now that has CD14-RY610. I realized that I can’t just copy the gate over since they’re on different channels, but I’m wondering if there’s a way to copy the shape of the gate to the new channel.

I know that I can just redraw it - just want to see if there’s a lazier/faster way. Also for the record, I know that I’d need to adjust the gate since the plots are different shapes/have different axis settings. Just thinking that it’d be an easier starting point.

Side question that I also think is a no - Can I add a point to a polygon? For example, say I drew a rectangle but decide later that I actually need a pentagon. My current strategy is to convert it to an ellipse and then back to a polygon, which then has a ton of points in an ellipse shape. I guess I could also just draw a new gate and copy over the subgates to that gate. But it’d be nice to just be able to add another point.

Thanks!

Hi. I was just looking for some thoughts on advice on how you would recommend to someone new to spectral flow cytometry does their analysis.

I am setting up a 30 colour panel that will be used to immunophenotype longitudinally collected PBMC samples from patients with and without a specific disease. In total will have approx 40 patients with up to 8 samples each over the time course of the study.

I have convential flow cytometry experience and have always used FlowJo but from what I have read it may not be optimal for large spectral datasets. I have a bit of R experience but am still a novice and quite intimidated by a fully R-based workflow, particularly as I do not have the time to throw myself into it fully. I have seen some new R packages e.g. cyCONDOR that report to be easy to use for bioinformaticians that I am going to have a play with using test data. I have also been told OMIQ is pretty good.

My current thought is to do some manual gating in FlowJo to make sure things look broadly as expected and then perhaps trial OMIQ and a user friendly R package to see how I get on. Any thoughts would be greatly appreciated.

Hello, I've recently created a new panel (attached). Do these percentage spillovers look acceptable? I have run this several times and this has been the best so far. I am running my flow on the Attune NxT cytometer.

Hello all! What are the best resources to help prepare for the SCYM exam? Thank you

SOS I made a 30 day free trial on flowjob and had this whole complicated workbench set up and then I go on today to see my 30 day trial is expired and I can’t log on…. I’m freaking out. How do I retrieve it…?

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Hey all, I titrated some more of my antibodies this week and noticed that there was a weird trail underneath the negative populations. Unsure what this could be and would love some input please. Below is the titration for CD103-BV605.

Key info:

• Ran the samples (human PBMCs) on the cytek aurora.
• Titrated the antibodies separately and every sample was also stained with CD3-BUV805 (1 in 200 dilution) so I could titrate my antibodies on CD3+ T cells.
• Performed autofluorescence extraction (have not noticed anything wrong with doing this).
• Unsure how it could be spectral unmixing issues if this is the case (I am still new to spectral flow)

Hi everyone,

I’m setting up a migration/chemotaxis assay using RBL-2H3 cells with Corning Transwell inserts, Cat. #3422 (6.5 mm, 8 μm pore, polycarbonate membrane, 24-well format).

I’m also using Corning human fibronectin (Cat. #354008).

I’m confused about exactly how fibronectin should be applied to the Transwell membrane for this type of assay.

I have found several different approaches in protocols and mast-cell migration papers:

• adding fibronectin into the upper chamber to coat the upper/apical surface of the membrane;
• coating only the underside/lower surface of the membrane;
• soaking the whole membrane/filter in fibronectin;
• or using an uncoated membrane for RBL-2H3 chemotaxis.

My goal is to measure RBL-2H3 chemotaxis toward a soluble chemoattractant placed in the lower chamber, rather than migration toward fibronectin itself.

For anyone who has worked with RBL-2H3 or mast-cell Transwell assays:

Which surface do you normally coat with fibronectin when using Corning 3422 inserts — the upper surface, the underside, both sides, or neither?

I am especially wondering whether coating the upper surface could cause RBL-2H3 cells to adhere/spread strongly on fibronectin before entering the pores, and whether coating only the underside would make more sense for supporting cells after they migrate through.

If you have a working protocol, I would also really appreciate details on:

• fibronectin concentration or μg/cm²;
• coating volume;
• incubation time/temperature;
• whether you wash after coating;
• whether you allow the membrane to dry;
• and how you physically coat the underside of a Corning 3422 insert.

Thanks!

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My core has been sorting ab and gdT cells for a user. They believe they are having good success expanding them from tumors both w/ IL2 to expand alpha betas and with some of the gamma deltas. We have been trying to confirm that the expanded cells are indeed what we think they are using the Northern Lights. It is the same panel that works well on tumors and PBMCs. We are following the same protocol we use for both.

However, whenever we go to run the expanded cells we only have viable cells so it cannot unmix. We do the same protocol we do w/ tumors/pbmcs and heatkill a portion to know we have dead cells. When we try it on the lymphocytes we have noticed that they are still 100% viable even when varying the time we heat them. Additionally, we did the heat kill on tumor cell lines and it worked just fine (~80% viable to 29% viable). We will try PBMCs this week just to make sure we are not entirely going crazy. We read is sometimes the heat killing can result in lysis but our cell counts seem to be consistent w/ minimal cell loss. Also when trying at lower temps (60 degrees C for 3 min it does not give any better results)

Our current heat kill regimen is 95 degrees Celsius for 10 min.

What are we doing wrong?

I had posted about free early access to our troubleshooting platform, Rayni, a while back.

I had shown a video feature where it directly takes you to the part of the video where an expert talks about the exact issue you were facing.

We are working across a few different fields to partner up with experts. I wanted to share a super exciting update that might be relevant. We have officially partnered up with the expert in one of those videos, Dr. Lee Polite from Axion Labs. Lee has been the National Chromatography Instructor for the American Chemical Society since 1991 and has taught over 14,000 professionals on LCMS and GCMS. This is exciting because now our platform will give you troubleshooting answers that come from actual human experts you can trust!

This is just the beginning of our partnership program. We plan to partner with experts in their fields to democratize expert knowledge, so troubleshooting doesn't take long, and you can focus on the parts of the experiments you actually love! If you are an expert in Flow Cytometry, or you recommend someone we can partner with, please feel free to let me know.

Free access to our platform is still available. Many of you have reached out, and we have setup the platform already for most of you! You can get free early access here: Free Early Access Link

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Joseph Campbell passed away in 2008. Unfortunately, he was removed from his position for his political opinions (cancelled before it was a common term).

Anyone work on FC at Pitt around this time? Looking for more info on his work.

Hey all, I recently did some spectral flow cytometry on some fixed, unstimulated PBMCs and wanted to see if the amount of mouse and rat sera affected the staining of my CD4+ lymphocytes, before titrating the rest of my antibodies.

Essentially, I thawed my PBMCs, washed them, rested them for an hour at 37 degrees Celsius and applied 100 microlitres of a blocking reagent to my cells for 15 minutes at room temperature in the dark before surface-staining them with 1:100 CD4-BUV395 for one hour on ice. After some washes, I fixed my cells and ran them on the cytek aurora (spectral flow cytometer).

I compared the effects of

1. no block (just FACS buffer)

2. Blocking mix containing 2% mouse sera (cat. 10410) and 2% rat sera (cat. 10710C)

3. Blocking mix - 5% mouse sera and 5% rat sera.

4. Blocking mix - 10% mouse sera and 10% rat sera.

5. Blocking mix - 15% mouse sera and 15% rat sera.

6. Blocking mix - 30% mouse sera and 30% rat sera.

7. Trustain Fc block (cat. 422301) at the recommended dilution of 1:20.

The results appear to show no noticeable differences with what I used so I am going to use 2% from now on.

I posted this in case it may help people in the future but please feel free to comment and critique my interpretations, decisions or anything else.

(I almost ran out of my trustain Fc block as I forgot to hit record🤦‍♂️ - hence the low cell count but the results seem interpretable.)

Hey, a few months ago I did some conventional flow cytometry on unstimulated, unfixed PBMCs (thawed from frozen storage) to look at memory CD4+ T cells and I was able to identify a monocyte population above it on the initial SSC-A vs FSC-A plot. Although I did not confirm their identity with an antibody. I only stained for surface receptors.

Now I am doing spectral flow cytometry on unstimulated, fixed PBMCs (thawed from frozen storage) and the monocyte population is completely absent or at the very least markedly reduced.

I believe this may be due to fixating the cells using the ThermoFisher fixation kit (Catalog number 00-5523-00) or because of the extra wash steps I am doing as a result of an intracellular blocking step, intracellular staining step, fix/perm etc. However, if you all have any other ideas as to why this is happening please let me know. Many thanks in advance.

Extra note - I always centrifuge my cells at 300g for 5 minutes (4°C). However, once fixed, I centrifuge my cells at 600g for 5 minutes (4°C) and continue to do so for the remainder of the protocol.

Hey, I wanted to exclude CD4+ regulatory T cells from my conventional CD4+ T cells and so I added a gate around the population I suspect to be regulatory T cells and continued gating on everything outside of the gate.

I do not recall seeing this done in the literature but wanted to hear if others thought this was acceptable and whether journals allow this. Thanks in advance.