Cell tak adhesive

Recombinant perfringolysin O was produced as described previously (Rossjohn et al., 1997 (link)). Viral particles purified by size exclusion chromatography were concentrated by 40-fold using Amicon Ultra-4 centrifugal ultrafiltration devices (Merck, UFC800324) and incubated with PFO at a final concentration of 0.5 μM at 37°C for 10 min in the presence of 0.1 mM inositol hexakisphosphate. A formvar/carbon grid (Ted Pella, 01801) was cleaned in a glow discharge unit and modified with Cell-Tak adhesive (0.05 mg mL⁻¹, Corning, 354240). The grid was then incubated with a sample (5 μL) containing PFO-treated viral particles for 1 min at room temperature, and the solution was wicked away using filter paper. The sample was immediately stained twice with uranyl formate (1% w/v) and excess solution wicked away with filter paper. The grids were dried under a gentle stream of nitrogen gas and allowed to dry further overnight. The samples were imaged using a FEI Tecnai G2 20 Transmission Electron Microscope equipped with a BM Eagle digital camera. Diameters of the PFO pores were measured using ImageJ.

The bioenergetics assay was performed using the Seahorse XF Cell Mito stress test kit and the Seahorse XFe96 analyzer, purchased from Agilent Technologies. The day before the assay, the tissue culture plate was coated using Corning Cell-Tak adhesive, according to the supplier’s recommendations. The Seahorse cartridge was hydrated with sterile water and incubated overnight in a CO₂-free incubator, at 37 °C. The day of the assay, the Seahorse medium was prepared, and the pH solution adjusted to 7.4. The following step involved seeding the cells on the previously coated culture plate. The cells were enumerated and centrifuged for 5 min at 2000 rpm, and the supernatant was removed and replaced with Seahorse medium. The cells were then incubated for 45 min in a CO₂-free incubator, at 37 °C. During the incubation, the stock solutions were reconstituted and diluted to the appropriate concentrations as follows: ATP synthase inhibitor oligomycin at 1.5 µmol/L, uncoupling agent carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) at 0.5 µmol/L and 2 µmol/L, fatty acid oxidation inhibitor etomoxir at 4 µmol/L, and inhibitors of complex I/III of the electron transport chain antimycin/rotenone at 0.5 µmol/L. The oxygen consumption rate (OCR) was measured at baseline and after sequential injections with the previously mentioned solutions.

Oxygen consumption was determined using the Seahorse XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). Briefly, after culture in LG, floating cells were collected and seeded onto wells of a dedicated 24-well XF24 cell culture plate previously coated with Corning Cell-Tak adhesive (Corning, New York, NY, USA), in unbuffered medium without glucose (complete formulation: XF unbuffered DMEM supplemented with 1% dialyzed FBS, 2mM Sodium Pyruvate and 2mM glutamine). After seeding, the cells were incubated for 40 minutes at 37°C without CO₂ before to run the experiment. H89 and FCCP were injected by the instrument.

Prior to the addition of CHO42 and CHO52, sterile circular coverslips were deposited into 24-well plates and coated with Corning Cell Tak Adhesive (at a concentration of 35 µg per ml, making sure the pH was in the range of 6.5–8). A 150 µl aliquot of a mid-exponential culture was added to the well. Following attachment, the cells were immediately fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton in 1× PBS. All primary and secondary antibodies used in the present study were diluted 1/100 in 1% goat serum in 1× PBS. Goat anti-rabbit IgG (whole molecule)–TRITC (tetramethyl rhodamine isothiocyanate) antibody and goat anti-mouse were purchased from Sigma–Aldrich. Coverslips were mounted on slides with Vectashield with or without DAPI (at a final concentration of 0.1 µg/ml).