Trypan Blue exclusion pipeline


My name is Fernanda and I am a research engineer at UCL. I’ve been trying to use Cell profiler to analyse my trypan blue exclusion pictures for three types of cells:

  1. Viable round cells
  2. Non-viable/dead (stained blue) cells
  3. Viable elongated cells

I am new to Cell profiler and by playing around with different pipelines I have managed to identify cells but I can seem to be able to differentiate from one type to another. Basically, I do not know how to tell Cell profile to pick those coloured cells as non-viable/dead. I haven’t got round to elongated yet. Any help I would appreciate. Attached a sample of one of my pictures.


This is the pipeline I have put together to identify viable and elongated cells, still struggling on dead cells. I can’t seem to get it to identify on a per object basis, those cells that are all dark (dead cells).

total_elongated_viable.cp (9.6 KB)

Hi Fernanda,

I’m attaching a pipeline that I think will get you closer to what you want (some tweaking is still needed). You already have most of the elements in place, I think. Some notes:

  • You can use the form factor as a measure to find elongated cells. Form factor is a value that ranges from 0 to 1, where 0 is a line and 1 is a perfect circle. Objects with lower form factors are more likely to be elongated. However, you will need to check the cutoff (which I set to 0.75 for the meantime) to see if it’s appropriate.
  • The viable cells are the ones that are left, i.e, not elongated with dark holes. If you change the cutoff in the first FilterObjects module, remember to change it accordingly in the second one.
  • Dead cells are the ones which do not have dark holes at all, which can be determined by find all objects, seeing which of the viable and elongated cells overlap with them and then filtering out those with overlap.

2013_04_01.cp (11.5 KB)

Mark, thank you very much for all the info. I will have a go at it this weekend and play around with it to see how it fits.

Once again, thank you



I’m also trying to get a Trypan Blue Exclusion pipeline for cell profiler, I can detect the cells (not all of them however) but cannot get the pipeline to differentiate between the blue stained, dead cells and the viable cells.

Any help would be appreciated, I am very new to cell profiler!

Thank you in advance,


Welcome! Could you please upload an example image and your current pipeline? That will be a good help for us to help you.


Attached are 2 pipelines I have been working with and a set of images!

Thank you,

<a class=“attachmExample Cell Counting.cpproj (409.3 KB)”
<a class="attachmExample Cell Counting 2.cpproj (409.3 KB)

Sorry for the long delay - I had started to work on this but brightfield images are often difficult. I’ll post my (unfinished) pipeline, but have you made progress in the meantime?

DL_Trypan.cppipe (11.2 KB)