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

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posted on 19.10.2015, 09:16 by David Mason, Joan Comenge, Raphael Levy

This data set is part of an open science project which aims at elucidating the fate and reliability of SmartFlares.

CELL CULTURE AND LABELLING

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.

CELL FIXATION AND PROCESSING

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.

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.

IMAGING

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.

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