Hello, my research team is using Cell Profiler to track the perinuclear localization of a drug into a cell. We are running confocal images, captured at a 20x magnification, with three different dyes through Cell Profiler.
We were hoping to get both perinuclear and peripheral fluorescence per cell and convert that data into violin plots. We are utilizing Cell Profiler to calculate the intensity in 5 bins within the cytoplasmic compartment to determine the perinuclear and peripheral localization of the specific drug. At the moment, we can only get cell fluorescence as an average per image, which encompasses approximately 10 to 20 cells. For the 100 cells we hope to capture for each data set, this would produce only 10 points for the violin plot, which is not enough.
Any details on how you are able to analyze per cell fluorescence with Cell Profiler would be extremely helpful. Any other advice for using Cell Profiler or further contacts who may have input on Cell Profiler would be appreciated. We thank you for any help you may give.
Blessings,
Conner Murphy
Dear Conner Murphy ,
first of all I would like to recomment the CellProfiler forum for specific problems. My experience there were great - a kind and responsive communtiy.
To come now to your problem. I would recomment to use a non specific stain for your cells. Drag5 might work to stain both cell body and nuclei. Otherwise you could use a CellTracker and Hoechst33342/DAPI to provide some spatial information of the cells. Than you should use identify primary objects to find your cells. (You should get now information for your staining per object (i.e. cell) Than I would use the DNA-staining to create a secondary object. These objects/masks can now be used to define the perinuclear space (by expanding and substration of the original mask).
Maybe you can post some sample images?
kind regards
Soenke
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