I'm working on 3D printed PLLA scaffold. I would like to see how well MG63 cells attach to the scaffold using confocal microscopy. My questions are,
1. Which dye should I use ?
2. Should I pre-label the cells?
3. Scaffolds are 2mm thick, will I be able to see them using confocal microscopy?
4. What should the time point to check the cell attachment and cell viability testing?
5. I will be using CCK8 kit for cell viability, if the scaffold absorbs the dye, does it affect my results?
Thank you,
Hi Divakar Karanth
You can use Hoechst 33258 dye (Invitrogen, Carlsbad, CA) to stain the nuclei,
to tests the cell adhesion, distribution, and proliferation of MG-63 cells
The fluorescent dye will excited at 350 nm and emitted at 460 nm. The blue fluorescence-stained nuclei can detecte by using a DAPI filter.
The distribution of cells with blue fluorescence-stained nuclei could observe and recorde using a spinning disk confocal microscope (Olympus CKX1).
I hope this could help you
1. If you are interested in cell attachment I'd go for a phalloidin/dapi staining, which will allow you to see the cell nuclei as well as the cytoskeleton. The staining is simple and the amount of information you can get from it goes beyond cell counting since you will be able to see their morphology.
2. There is no need to pre-label the cells. You can stain them after adhesion, (fixing them at this point would also be a good option).
3. If your cells will be attaching to the surface of the scaffold, you should be able to see them. However, if there is porosity and they go inside the scaffold it may be hard to trace those cells right away, but you will always be able to see the outer surfaces.
4. For adhesion tests, 24 to 48h are a good option. For viability it depends on how long you want to track the response of your cells.
5. I've never worked with CCK8. If it would be sequestered by the material then yes it could affect the results but you should be able to spot it and eventually change the dye. Anyway I don't see how it could be uptaken by the material to such an extent.
Cheers
As the answer above, hoechst and DAPI are both great selection for nuclei.
If you're interested in cell adhesion properties, E-cadherin could be a marker for you to analyze. I think it would be more representative than staining cytoskeleton.
You should set a blank about CCK8 kit and your scaffold only to deduct the background OD value.
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