# Deposition of Cell Culture Coatings Using a Cold Plasma Deposition Method
## 2 Materials and Methods
### Plasma Deposition of Collagen Film
_Microscopy._ Scanning Electron Microscopy (SEM) images were taken using a Hitachi S300N SEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 5-10 kV and a probe current of 35 mA. The samples were mounted on a high purity aluminum slab using conductive carbon tape. Atomic Force Microscopy (AFM) images were taken using a Bruker Dimension 3100 AFM (Bruker, Karlsruhe, Germany), at room temperature, using an Adama Diamond tip. The data acquired was analyzed using Gwyddion software. _Surface Energy._ Contact angle measurements were conducted using a Kruss TVA100 (Hamburg, Germany) which uses a top view method to determine contact angle measurements and facilitated direct measurements of contact angle within the well of a microplate. Measurements were taken using a 1 uL water or hexadecane droplet taken at equilibrium, 60 s after placing the droplet on the surface and surface energy was then determined using the method of [PERSON] and Wendt [26]. _Gel Electrophoresis._ The SDS-PAGE analysis was performed using 4-12% hand-cast gels. Protein concentrations of all samples were adjusted to 1 mg/mL by mixing with loading buffer (2.5% of sodium dodecyl sulphate (SDS), 10 mM of Tris-HCl, 1 mM of EDTA, and 0.002% of bromophenol blue) with 5% of \(\beta\)-mercaptoethanol, used as a reducing agent to break down the disulfide bonds in the proteins. All samples were heated for 5 min at 100 \({}^{\circ}\)C before electrophoresis. Then, 10 uL samples and a marker were added to the wells in the gel. The gels were run at 200 V for 1 h and stained with Coomassieie brilliant blue overnight. The gel was destained three times using 10% acetic acid solution washes over a 24 h period. Gel imaging was performed using G Box Chemi XRQ (Syngene, Cambridge, UK) for visual analysis. _MTT Cell Culture Assay._ The human hepatoma (HepG2-ATCC) cell line and the human epithelial carcinoma (HeLa) cell line were obtained from the laboratory of Prof. [PERSON] at Dublin City University. A cryostat of each cell line was thawed at 37 \({}^{\circ}\)C and resuspended in prewarmed cDMEM (Dulbecco's Modified Eagle Media; 10% (v/v) Fetal Bovine Serum; 1% (_w/v_) Penicillin-Streptomycin). Cells were centrifuged (1200 rpm; 3 min), the pellet resuspended in 10 mL of cDMEM (37 \({}^{\circ}\)C), added to a T25 cell culture flask and incubated overnight in a humidified atmosphere (37 \({}^{\circ}\)C; 5% CO\({}_{2}\)). Cell growth was monitored by examining cells under a Nikon Eclipse E400 Microscope. After 24 h of growth, cells were subcultured into a T75 cell culture flask and a subculture routine was implemented to ensure optimal cell growth conditions were maintained. When HepG2 and HeLa cells reached 75%confluency, media was removed from the cell culture flasks, cells were rinsed with sterile Dulbecco's PBS and incubated with 2 mL of 0.5% (_w/v_) Trypsin (37 \({}^{\circ}\)C; 5% CO\({}_{2}\); 5 min) until they were no longer adherent to the flask. cDMEM was added to inactivate the trypsin and the cell suspension was centrifuged (1200 rpm; 5 min). Cells were resuspended in 1 mL cDMEM. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide) assay ab211091 (Abcam, Cambridge, UK) was used to assess the metabolic activity of the cells when coated on the various 6-well cell culture plates. The assay was carried out according to the manufacturer's guidelines. Briefly, HepG2 cells were plated at a density of \(7.5\times 10^{5}\) cells/mL and 2 mL of cell suspension was added to each well. HeLa cells were plated at a density of \(5\times 10^{5}\) cells/mL and 2 mL of cell suspension was added to each well of the 6-well culture plate. Cells were grown for 24 h (37 \({}^{\circ}\)C; 5% CO\({}_{2}\)) before supernatants were removed and serum free media was added (500 \(\upmu\)L/well). MTT reagent was added to each well (500 \(\upmu\)L/well) and the plate was incubated for 2-3 h (37 \({}^{\circ}\)C; 5% CO\({}_{2}\)). MTT solvent (1.5 mL) was added to each well, sealed, and placed on the orbital shaker for 15 min.