Uptake of SmartFlare (Spherical Nucleic Acids) in HeLa cells: Transmission Electron Microscopy results

2015-10-19T09:16:24Z (GMT) by David Mason Joan Comenge Raphael Levy
<p>This data set is part of an open science project which aims at elucidating the fate and reliability of SmartFlares.</p> <p>CELL CULTURE AND LABELLING</p> <p>HeLa cells were seeded at 500,000 cells per well of a 35mm polystyrene dish.The Cy3-VEGF SmartFlare was reconstituted into 1 mL of nuclease free water. 20 μL of the Cy3-VEGF SmartFlare was added to 980 μL MEM medium, which also contains Fetal Bovine Serum (FBS), non-essential amino acids and 1% penicillin and streptomycin.Existing medium was removed from the settled cells and a 2 mL PBS wash was carried out twice. Once all PBS was removed 1 mL of the SmartFlare/medium mixture was added to the cells. The cells were then placed back into a 37°C, 5% CO2 incubator for 18 hours.</p> <p>CELL FIXATION AND PROCESSING</p> <p>After 18h incubation, cells were fixed with a solution containing 1% paraformaldehyde and 3% gluteraldehyde in 0.1 M cacodylate buffer (pH 7.4). They were stained first with reduced osmium (2% OsO4 + 1.5% K4[Fe(CN)6]). This was followed by a 2nd osmium staining (2% OsO4) and a uranyl acetate (1%) staining. Samples were then dehydrated in graded ethanol (30%, 50%, 70%, 90% and 2x 100%). Finally, samples were infiltrated with medium TAAB resin 812 and embedded with the same resin. The resin was cured for 48h at 60°C.</p> <p>Ultrathin sections of 350μm x 350μm x 74 nm were cut and placed in 200 mesh Formvar/Carbon filmed grids. They were post-stained with uranyl acetate and lead citrate before TEM imaging.</p> <p>IMAGING</p> <p>Cells were imaged on an Tecnai G3 spirit. Cells were imaged first at low magnification (~2900x) and also at higher (~6800x) magnification in the same area.</p>