Hi Rachel Burke

I don't know of any widely used programs specifically designed to measure confluency in suspension mammalian cells from microscopy images, largely because, as you stated, suspension cells are free-floating and exist at different focal planes, making image-based analysis more complex than for adherent cells. In suspension culture, confluency refers to cell density within the medium, not surface coverage, and is typically measured through cell counting, optical density (OD) or viability staining rather than just microscopy images. In our lab, we typically just use Trypan Blue with a hemocytometer to assess cell viability and density. With experience, you can also visually detect media acidification by noting changes in the pH indicator’s color.

However, MAYBE tools like CellProfiler (with 3D/z-stack support), Aphelion, and commercial platforms like Cell3iMager or CloneSelect can be adapted to segment and quantify cell clusters from z-stacked or focused images but I don't think it will be a worthwhile investment.... depends on your experimental idea, I guess.

All the best!

Rachel Burke , hello.

At present, most of the automatic confluence analysis software on the market (such as Cell Confluency Tool, SnapCyte, etc.) are mainly designed for adherent cells, because adherent cells are attached to the same plane, and it is easy to calculate the confluence. However, since suspended cells are distributed in different optical planes, the method of directly measuring the coverage area is not applicable.

I also thought that perhaps the total area covered by cells/colonies can be calculated and divided by the total field of view area to obtain the "three-dimensional projection confluence". However, there must be errors. If you are used to using software such as ImageJ/Fiji, you can also do automatic segmentation (such as threshold, edge detection, deep learning model segmentation), which can reduce some errors.

If you need detailed ImageJ/Fiji operation procedures, please contact me for further information.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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Hi Rachel,

You could try using our Phoretix Colony Counter software - https://totallab.com/software/colony-cell-counting-software/ however it's not compatible with a full z-stack of images so it would be layers by layer and image by image, quite manual I'm afraid but the automatic colony detection should be able to pick out your cells quite nicely.

You can give it a try for free on our website so no risk to you in it not working.

Best wishes,

Steven

I agree with Lucian, our lab uses SnapCyte for our cell suspension count, and it works pretty well. Try them out. I also think they are free to use if you have an academic email account.

I wanted to add on top of Lucian Miles comment. I totally agree that tools like SnapCyte can analyze cell count and confluency mostly in adherent cells, however... SnapCyte can also accurately analyze hemocytometer images. Therefore, you can load your suspension cells in the hemocytometer and measure the count using tools like SnapCyte, resolving the overlapping issue.

Rachel, Firstable, I think you can resolve it by photomicrographs with DIC microscope where you can determinate one focal plane (confluence cells) after it you can determinate region area of these cells by imageJ. First resolve it with your microscope before think in software :) Rachel Burke