r/flowcytometry

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Hey everyone,

I'm having some trouble approaching my gating to get the "clean events".

Specifically, aside from clear debris (low SSC/FSC, non-specific binding), I have one population that is slightly lower than lymphocytes on FSC, about the same on SSC. I am expecting a lot of immature cells in my samples (lower FSC), but also there is a lot of dead cells (also expected). When plotted against viability, this lower FSC population sometimes shows increase in viabilty dye binding, with a specific population in what i would consider "dead" cells region - but not always! Some plots as an example (singlets removed, gated on CD45+ events). This is consistent across all my samples, regardless of sample quality (as you can see, it's quite variable).

I could use some advice on how to approach this. My current train of thought is to assume that it is immature cells and include them in the lymphocyte gate, but exclude this population in the dead cell region with the assumption that it's early apoptotic cells (keep the boundary, whether it is there as a clear population or not).

Thanks in advance!

Hi

I am trying different whole blood lysis buffers to identify the most suitable one for our application. Below are a few flow plots generated using lysis buffers from different vendors (BD, BioLegend, and eBioscience; not shown in the same order). The samples were washed after lysis, and only the second sample was fixed; the others were processed using lyse/wash only.

I worked with whole blood about 10 years ago, but I do not remember seeing a split or double granulocyte population. Is it normal to observe two granulocyte populations after lysis? Is it like mature and immature granulocytes ?

https://preview.redd.it/xym51mz3sc2h1.png?width=2102&format=png&auto=webp&s=48d1d9d376abad136a5a1208abbd43843145f6d8

Hi everyone!

I'm having some curious issues with the downsample plugin on FlowJo 10.10.1.

I need to downsample CD4+orCD8+ population from fcs files across 3 groups (spleen, blood and tumor). When I apply it on blood and spleen groups, it works well producing the downsampled population for each fcs file and with the correct number of events (1262 based on sample with less events).

When I run it on the tumor group it works only on some fcs files and produces downsampled population with a variable number of events different from what I select.

How to resolve it? I restart the program hundreds of times

Hi all,

I’m new to the CytoFlex LX and would like to share my observations and get some advice from this community. My LX is equipped with the deep well plate loader module.

1. When running samples (single stained comps for now) using standard 96-well v bottom plate I see this strange phenomenon where signal from the previous well is contaminating the next sample but that signal gradually goes away with time (number of events go substantially down after about 10-20 seconds). I use 1.5 sec mix and 5 second backflush. I should mention that the event rate initially is also somewhat higher than later but the loss in contaminating events is still greater given the percent of contaminating events per a time interval goes down with time if that makes sense (almost ~15 % during first 15 seconds, then ~5% during next 15 seconds, then ~0.5% during the next 30 seconds at Fast Flow Rate ). Do I need to make backflush even longer?

2. When going from Sample Flow Rate Slow (10uL/min), to Medium (30uL/min), to Fast (60uL/min) I see very noticeable broadening of signal in pretty much every channel including scatter. Is this me or is this particularly true for this specific platform? I don’t recall this with BD instruments (Fortessa) when going between flow rates. I believe others observe this. What is your recommended sample acquisition settings then? Concentrated samples (50uL instead of 200uL per well final resuspension in plate?) acquired slow?

3. On a Time vs Fluorescence or Scatter gate, I see what appears to be a gap of about 1 second in data every about 10 seconds. I assume this is the peristaltic pump. I thought swapping the peristaltic sample line would alleviate this but it did not. Is this normal behavior then?

Most concerning to me is #1 as I was expecting carryover to be <0.5% according to the spec of this instrument. I appreciate everyone’s input.

did some testing using stored cells for fixable dye live-dead-stain, just want to share it here.

https://preview.redd.it/1u55milpn02h1.jpg?width=791&format=pjpg&auto=webp&s=14fc7fcad8e880230f72ba26462d7158608693d4

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Hiya,

I'm really new to flow cytometry and wanted some help with normalizing and representing my cell counts. I stained my cells with PI and now my supervisor wants me to plot the cell counts of PI-positive cells to show %killing. However, I'm not sure what formula I should use to normalize these values? My supervisor said to transform counts into percentages- by dividing- Experimental cell count / control cell count *100. But in my head, this works if cells were live and the control was normalised to 100. For %killing, the control becomes 0. So, how would this work? I would have a fold change of over 200 (eg. if experimental cell count was 32,070 and control was 13,089)?

I'd appreciate any help I can get!

Simple question, anyone detected this fluorescence protein before? I got a plasmid that should constitutively express mIFP, but after transfection, no fluorescence can be detected in all channels in the far red laser in bd symphony s5 ae. The same cells has been transfected with gfp and cfp with high transfection rate. Plasmid sequenced no problem…

Hello, flow cytometer users!

Our lab recently got a Cytek Aurora and we are pretty much happy about it so far.

In terms of maintenance, the technician from Cytek ask us to perform QC beads running everyday prior to any measurement of the day.

But, I am genuinely curious if I can save some beads by doing the QC procedures maybe weekly.

It is partly because, in my previous workplace, I had a weekly beads-based calibration for BD flow cytometers.

If you're in the lab with Cytek Aurora, how do you guys maintain the instrument?

Do you run QC beads everyday?

If you have any suggestion, that would be wonderful!

hi! so i’m doing Tfh-B cell co culture. One of my gates would be CD4 vs CD19.

I currently have BV650 for CD19 in lab and a few CD4: options available: BV605, AF700, APC.

I was leaning towards CD4(AF700) and CD19 (BV650) and was wondering if this would provide the most efficient separation for clean downstream gating? I’m on a spectral cytometer and any insight would be appreciated! Thanks!

Hello,

I was wondering if anyone knows the percentage for gating "resting" cells. I have seen a lot of people mention the 2% and even 5%, but I want to know if there is anything backed by literature about the gating?

TIA

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!

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.

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.

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!

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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!