I know that it would be better to stain cell nuclei with DAPI. However, when I take the images, I have a strong background of fluorescence, probably due to the use of cotton swab tfor remove cells in the upper nside of the transwell.
It may was air bubble under coverslipes. Make sure there is not any air bubbles between slide and coverslipes. could you attach the image here?
I used hematoxyline stain and it was easy for me. I counted them manually and was not very difficult.
It is a bit hard to judge what would be the best way without an example image (could you add one?) See also: http://fiji.sc/Particle_Analysis
If there are a few nuclei per image or if this is a once-off analysis, I would agree with Moumita, use the Cell Counter plugin. It assigns a marker on the particle/nuclei counted which minimizes the error associated with the analysis.
When able to physically separate the migrated cells from less migrated cells (as you have done using a transwell type assay) then simply extract the crystal violet and quantify using spec. Alternatively PicoGreen would be a more direct measure (based on dsDNA extracted. Obviously do this after removing the cells on upper surface with cotton bud.
- Badges
- Science topic
- Similar topics
- Medicine
- Physiology
- Biosignals
- Biomedical Signal Processing
- Statistical Analysis