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Cell Assay Protocol 002: Confocal Microscopy to Test the Internalization of RNA Nanoparticles within Cells

1. Culture cells on cover glass in their complete medium overnight.
2. Wash cells in blank medium twice before experiment. Incubate cells in each well with fluorescent RNA nanoparticles at a final concentration of 50 nM to 200 nM at 37 °C for 1 to 4 hours; wash cells with PBS.
3. Fix cells with 4% paraformaldehyde at room temperature for 20 minutes, then wash cells with PBS.
4. Stain cells with a cytoskeleton staining reagent or membrane staining reagent, then wash cells with PBS.
5. Mount the cover glass with cells onto a glass slides, and stain the cell nuclei with DAPI. After drying, the slides are ready for confocal microscopy analysis. Choose excitation and emission spectrum based on the information from your product.
 
Reference:
1. Haque F, Shu D, Shu Y, Shlyakhtenko L, Rychahou P, Evers M, Guo P. Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today. 2012; 7(4):245-57.
2. Khisamutdinov EF, Li H, Jasinski DL, Chen J, Fu J, Guo P. Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles. Nucleic Acids Research 2014; 42(15):9996-10004.
3. Pi F, Zhang H, Li H, Thiviyanathan V, Gorenstein DG, Sood AK, Guo P. RNA Nanoparticles Harboring Annexin A2 Aptamer Can Target Ovarian Cancer for Tumor-Specific Doxorubicin Delivery. Nanomedicine 2016 Nov 25. doi: 10.1016/j.nano.2016.11.015.
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