I’m new with Cell Profiler and I need to build a pipeline to measure transfection efficiency with GFP in HEK293T cells.
I am using lipofectamine to insert a vector with GFP in my cells and I also use Hoechst for nuclear staining. I want to measure the % of cells that present both blue and green fluorescence compared to the GFP negative nuclei. Here is an overview of my pipeline:
- IdentifyPrimaryObjects identify nuclei from the nuclear stain image (Hoechst staining images).
- IdentifySecondaryObjects identify cells stained with GFP (GFP staining images)
- MeasureObjectIntensity determines whether a nucleus is GFP-positive by measuring the nucleus intensity from the GFP channel.
- DisplayDataOnImage displays the per-nuclei median intensities overlaid on the nuclei to visually determine a cutoff for GFP positive/negative.
- ClassifyObjects classifies the nuclei but on the basis of the median intensity measurement.
- FilterObjects filters the nuclei on the basis of median intensity measurement or other parameters.
- CalculateMath to obtain a per-image % of GFP-positive nuclei: divide the GFP-nuclei count by the total nuclei count and multiply by 100.
I read the tutorials and used some of the tips that I have found in this forum but I still have doubts about the accuracy of results…I am concerned about segmentation issues and the detection threshold for measuring fluorescence intensity… any tips to improve the quality of images and the segmentation? Am I using the right approach? Also, I was wondering if it would be possible to use the bright field image for cell counting?
I am attaching my pipeline and some sample images here. Open to suggestions!
ValidatedPipeline.cpproj (767.3 KB)
Thank you very much.