r/flowcytometry

Hi all,

Looking for some guidance here. I have some stimulations I need to do on Rorgt-eGFP positive cells (mice). The ideal situation for me would be to stimulate all leukocytes and run intracellular cytokine staining for IL-22.

Of course with GFP permeabilisation becomes difficult. I was wondering if anyone had any experience in this or has done something similar before? Has anyone tried this using the BD cytoperm/cytofix kit? It’s a transcription factor so I feel it might be okay relative to a cytosolic protein.

My backup plan is to sort but these cells are so rare I think this will be difficult.

Thanks!

Has anyone successfully stained reference controls in something like a 96-well plate instead of tubes for higher parameter panels?

I’m currently running 13–20 color panels on our BD A8, stain my primary samples in 96-well plates and my reference controls separately in tubes simultaneously during the same protocol. However, the mix of both substantially slows down prep, especially when processing multiple tissue types or staining multiple panels.

As I'm building higher parameter panels, I’m considering staining the reference controls in plates as well. My main concern is potential cross-well contamination/spillover during wash steps (ex from flicking) subtly altering the spectra and impact unmixing accuracy. I have tried this with some success in smaller panels, but as I develop higher parameter panels I'm a bit paranoid.

Any advice/input is appreciate. I'm curious if anyone has any workflows that have worked well for them.

Thank you!

Yol know any resources where I can theoretically study about flow cytometry - protocols, uses, optimisations and troubleshooting at a beginner to moderate level?

I wanna have as much information as possible before I get into my job.

We’d be using flow cytometry during car - T manufacturing.

My lab is trying to recover B cells from human spleen, and it's been really tough to get cells in good condition. This may be because of delays in tissue isolation and shipping post mortem. After isolation we have barely got any cells that look intact (based on live/dead staining and flow).

Does anyone have lessons learned, or recommendations for cell dissociation and recovery methods? We are primarily looking to use the cells in flow and RNA-Seq.

Does anyone know how to show cells/uL for a stats output from a Cytek FCS file? On the cytek PDF it shows easily, but I am doing all processing on Flowjo.

Hi! I am quite new to flow cytometry and need to titrate a new antibody for our panel. From what I've read, it seems important to titrate on the same cell numbers one would have in the real samples for representative level of antigen saturation. Would it be incorrect to use much smaller cell numbers, in the interest of saving samples? We usually don't use a set number of cells for flow either, but run the whole samples, which can differ in number of cells.

Apologies if this has been asked already in the subreddit. Many thanks!

We're looking to acquire a spectral cell sorter, for a clinical/academic setting that has full time technical staff, but who would be across this and other non-flow instruments. We're considering the Sony FP7000, Cytek Aurora CS and BD facsdiscover S8.

Of these, would any be operable for sorting without hands-on intervention by full-time staff? And related to that, would they perform well as analysers for users who don't need sorting capabilities?

Thanks!

Would you use as unmixing control matching fluorochrome but all in CD4 or CD45 or you would always need to match the conjugated you are using? How often would you do the unmixing when you keep running the same panel?

Has anyone ever seen such crazy autofluroescence as these unstained microglia? I'm trying to design a 9 color panel but feel like this might be impossible with these cells!

https://preview.redd.it/cohm7vxh4yzg1.jpg?width=2136&format=pjpg&auto=webp&s=d7eae070ba356bcb6e8873b5bef1e811458acca8

hi! I have a few questions about panel design and would appreciate any insight! thank you so much!

1. I currently use PE/Cy7 (CXCR5) and PE/Dazzle 594 (BCL6) in the same panel and it’s worked great. I wanted to add IL-21 (PE) in since we have that in lab but I believe that’s not ideal??? I’m considering APC instead??

2. BV605 and Pe/Dazzle 594 overlap but since they’re excited by different lasers would it be fine?

3. Does anyone have any experience with BV421 (FOXP3) or have any recommendations for any other bright antibodies other than PE?

thank you again!

Hello all,

As the title states, we've run into an unfortunate problem with our Flow Cytometer. We're using a pretty old model (as far as my Pi has told me, it's over 20 years old). It hasn't been used for a few years, and when we've tried, we keep running into an error telling us that the cytometer status is not 'ready'.
I worked with the lab manager trying to find sources for the problem, and the first thing we're running into is corrupted calibration files. It won't pick up anything from a blank. Since it's an older model, BD no longer supplies a manual or has any people to send for repair.
Would anyone know how I can locate replacement calibration files, or what we could possibly do past this? We were really hoping to get ours working so I could get some training in and create a protocol for my research project.

I performed spectral flow cytometry on a Cytek Northern Lights, but a few samples appear to have taken up bubbles during acquisition. I later realised some wells likely had <100 µL volume due to uncalibrated pipettes, so I suspect the instability is from low volume/bubbles during uptake.

The issue is that the affected samples/reference controls contain obvious unstable acquisition periods that I want to remove before unmixing.

What I’ve tried so far:

1. In SpectroFlo, I can create a time gate (P2) before unmixing, but during unmixing, it still seems to reference the original ungated events rather than the P2 time-gated population. I also can’t seem to change the channel/source population used for unmixing.

2. I then exported the raw FCS files into FlowJo, applied time gating there to remove the unstable regions, and exported the gated populations as new FCS3 files with all uncompensated parameters.

3. However, when I try to re-import these gated FCS files back into SpectroFlo for unmixing, I get “Possible file corruption or wrong files uploaded”.

I suspect this may be due to SpectroFlo/Cytek metadata being altered during FlowJo export, even though the files open fine in FlowJo.

Has anyone dealt with this workflow before on Cytek instruments?

1. Is there a proper way to time gate/remove bubbles BEFORE unmixing in SpectroFlo?

2. Can SpectroFlo unmix from a gated population rather than the parent acquisition?

3. Is there a known method for exporting FlowJo-gated Cytek files back into SpectroFlo without triggering file corruption errors?

Any advice would be massively appreciated  🙏🏼