Hello Expert Users,
I will appreciate if you could tell me the different steps to count positive GFP cells in transfection experiment and non-transfected cells to calculate transfection efficiency
Thanks in advance
Hello Expert Users,
Which software suite you want to use will likely depend on how many images you’d like to quantify, but here is the general approach for this sort of assay:
- Identify and segment nuclei (example tutorial here).
- Associate cytoplasm with each nucleus (you could just expand each nucleus by x pixels if you only need to know a value for positive vs negative).
- Quantify average intensity of GFP per cell.
- Define an average intensity cutoff for when cells will be considered ‘positive’.
- Transfection efficiency = positive cells / total cells.
Hope that helps!
If the nuclear and cytoplasmic regions are binary, one nice way to get this resolved is using the Region Connection Calculus.
I have to perform a theoretical approach to the use of CellProfiler. I have (theoretically) performed a transfection of a plasmid coding for a fluorescent protein (GFP). I stained the nuclei with DAPI. Could you explain to me how to measure the efficiency of the transfection? (in a theoretical but detailed way since I can’t do it in real life because as i said, it’s a theoretical approach). @DStirling I have figured out how to segment the nuclei with ImageJ but what to do next? And what is the link between ImageJ and CellProfiler? I’m sorry if my questions are not clear but I’ve only known about this software for 30 minutes.
@sarah_g - No problem! CellProfiler and ImageJ are separate software packages. They’re not easily linked together if you’ve not got experience with both.
Speaking in general terms, ImageJ excels at providing a toolbox for manipulating individual images and analysing them. CellProfiler is aimed at automating analysis of multiple images using a ‘pipeline’ of analysis operations. You can automate ImageJ as well, but this takes some knowledge of scripting.
It sounds like the analysis you desire is relatively straightforward. In CellProfiler you could probably do this by combining the IdentifyPrimaryObjects, IdentifySecondaryObjects and MeasureObjectIntensity modules in a pipeline. With ImageJ, if you’ve already managed to segment the cells, you should look into the Analyse–>Measure menu.
@DStirling Thank you very much for your response. My PC doesn’t support the installation of CellProfiler (and because of the confinement I can’t go to university at the moment (belgium)) so is there a tutorial with these different steps (including how to separate two cells that are touching each other)? I need to detail how to calculate the efficiency of a transfection, as if I was doing it, but without doing it. I know it’s a rather special job, but I need a protocol that describes step-by-step how to do it. Is there such a thing?
Thank you very much!
I’m surprised that you can’t install CellProfiler, but if that’s the case then ImageJ looks like the best way to do this. This tutorial may help you.