Co-culture Pipeline check

pipeline
correlation
doseresponse
coculture

#1

Hi,

I have been trying to compare the mean intensity of the xeno (small circular cells) created in ilastik/CP and % of viability by flow and see if the correlation between these 2 measurement. So far the R value is very low and I’m not sure if I have missed anything along the way. I’m wondering if anyone can have a look at my pipeline and give me some advice?

many thanks!
shu

290517_coculture probablities import v4.cpproj (823.0 KB)

it is a 5pt dose of ABT263, 5 fields/well, 1:4 , starting @ 0.1uM

Flow results of ABT263 is as this graph


#2

Your pipeline seems to be doing reasonable things, though I’m not really sure on the biology so I can’t say for sure.

I think if you’re not getting the results you expect you need to

  1. Make sure you agree with the probability maps you’re getting from Ilastik
  2. Make sure you agree with the CellProfiler segmentations of your cells (is it finding the right cells, are some getting missed, etc)- I often find adding an OverlayOutlines+SaveImages modules is nice at this stage so you can quickly flip through the segmentations.
  3. Think about whether or not mean intensity is the right metric to judge viability- again, without knowing the background of the experiment or even what dye you’re using it’s hard for me to say.

You could also think about using CellProfilerAnalyst to look at your data; you could add the dose information in the Metadata module, export the measurements to a database and then use the graphing tools, plate viewer, etc to see if you can find something in the CP measurements that nicely correlates with the dose-response that you expect.

Good luck!


#3

Thank you Beth for looking into my pipeline. I really appreciate it.

I did check the probably map from ilastik and seem fairly accurate. In terms of CP, I would try overlay outlines + save images module as you have suggested. Thanks for your tip!

In brief, I’m trying to establish a co-culture system whereby the xenograft are supported by MSCs. In the past, we observed the monoculture won’t survive the drug treatment thus there was no meaningful results. So in this co-culture system, we are hoping to do have a viability readout in a high throughput imaging manner.

We have been trying to use a dye called CyQUANT which stains the viable cells (https://www.thermofisher.com/au/en/home/brands/molecular-probes/key-molecular-probes-products/cyquant-cell-proliferation-assays.html). The reason why I’m looking at mean Intensity is because CyQUANT tends to have a gradient of signal. It composes of a fluorescent dye which binds to DNA and a suppressor which is permeable to leaky cell membrane, binds to CyQUANT and suppresses it from fluorescencing. The amount of inhibition also depends on how leaky of the membrane is and how much suppressor is present, and the position of the DNA. I spoke to the company technical support and confirmed this and thus the dye is not really as simple as “on or off”. What do you think about this? Have you come across any experiment which uses dye with similar mechanism?

As for adding the dose information in the metadata module, would you be able to explain a bit more? Sorry I’m still quite new to imaging analysis…

many thanks!
shu


#4

You may want to check out the Translocation tutorial; it’s a good intro to CP in general and also includes instructions and examples for adding dose metadata to a pipeline.

I’d consider staining your cells with DAPI and this CyQUANT dye, then identifying nuclei using the DAPI channel and measuring them in CyQUANT- otherwise for nuclei that are leaky and therefore are super dim in the CyQUANT channel, you’ll never actually find them. This might explain why your data don’t correspond with what you’re seeing by flow- the actual leaky cells are totally missing if you’re identifying based on CyQUANT.


#5

Thanks Beth. I will try your suggestion!