I’m working with a mammalian cell culture system in which i’ve induced cell death and am trying to sort out the mechanism. I’ve done TUNEL staining to look at endpoint apoptosis. The kit is from Roche and stains TUNEL positive nuclei green (FITC). I’ve also stained nuclei with DAPI. The images load as greyscale images in cellprofiler.
My goal is to quantify the difference in TUNEL fluorescence activity between samples - the image acquisition was via a fluorescence microscope. A post-doc in my lab turned me onto this program after I had little sucess with image-pro plus.
I have a basic understanding of this program, but using a sample (human cells from the website) pipeline in which they first isolate the nuclei by way of the DAPI image in the green image, and then apply the objectintensity module - to my images yields inconsistent and confusing results. In images with little intensity, i get higher numerical values for the intensity vs. images in which I have strong green signal. I suspect that the background, which is “black”, but is likely influencing the quantification and producing the inconsistencies i’m seeing. I’m trying to build a module to isolate the green nuclei (it is crucial that i ignore any cytoplasmic green signal) and then eliminate the “background” before quantifying it and i’ve had little success in doing so. Has anyone had any success with trying to build such a module, or is this application not suited for cellprofiler?
Thanks for all the help!
p.s. i can upload the images if necessary - just ask