8 well glass chamber slides

Cells were grown adherent on 8-well glass chamber slides (Nalge Nunc International, New York, NY, USA). The IF was performed as described [23 (link)]. For analysis of phosphorylated paxillin, cells were analyzed after adhesion on fibronectin or collagen I. Samples were analyzed using an Eclipse TE2000-S microscope with a 40× 0.75NA PanFluor objective (Nikon, Tokyo, Japan). Images were acquired with ACT-1 software (Nikon) and processed using ImageJ and Adobe Photoshop softwares. Confocal microscopy was carried out using a Leica TCS SP8 X confocal laser scanning microscope (Leica Microsystems GmbH, Mannheim, Germany). Images were acquired in the scan format 1024 × 1024 pixels in a single plane using a HC PL APO CS2 60×/1.30 oil-immersion objective and a pinhole always set to 1 Airy unit and analyzed using Leica LAS AF rel. 3.3 (Leica Microsystems GmbH) software. Images were processed using ImageJ and Adobe Photoshop software.

EOC MCAs, released from the scaffold as described above, as well as cells grown adherent on 8-well glass chamber slides (Nalge Nunc International NY, USA), were fixed with 2% paraformaldehyde for 20 min and permeabilized for 10 min in PBS containing 0.1% Tween 20. For the immune reaction with anti-β-catenin Ab, cells were fixed with methanol for 10 min. IF detection of E-cadherin on cell lines was routinely performed on confluent cells to be sure to visualize stable cell-cell contacts. Indeed, in confluent monolayers, some SKOV3 cells also display E-cadherin expression. Samples were analyzed using an Eclipse TE2000-S microscope with a 40X 0.75NA PanFluor objective (Nikon, Tokyo, Japan). Images were acquired with ACT-1 software (Nikon). Confocal microscopy was carried out using a Leica TCS SP8 X confocal laser scanning microscope (Leica Microsystems GmbH, Mannheim, Germany). Images were acquired in the scan format 512 × 512 pixels in a single plane using a HC PL APO CS2 40X/1.30 oil-immersion objective and a pinhole always set to 1 Airy unit and analyzed using Leica LAS AF rel. 3.3 (Leica Microsystems GmbH) software. Images were processed using ImageJ and Adobe Photoshop softwares.

BMDM (2 x 10⁴ cells/well) were cultured overnight on 8 well glass chamber slides (Nunc Inc, IL, USA) in DMEM with 10% FCS. Cells were treated with FITC-polyI:C (2.5μg/ml) in Optimem media (Life Technologies) with or without lipofectamine 2000 (Invitrogen, at 1:100 dilution, 10μl/ml) in the presence or absence of hBD3 or TAMRA-labelled HBD3 (0.5μg/ml). After 2, 10, 15 or 30 mins cells were washed in PBS and fixed in 4% PFA. For early endosome staining, cells were blocked with 10% donkey serum and incubated with anti-EEA11 antibody (Abcam, UK) for 1 hr at RT, then 30 min with donkey anti-rabbit Cy5. Cells were imaged using a 40x 1.3NA oil immersion objective on a Nikon A1R confocal microscope using Nikon Nis-Elements AR software for image acquisition (Nikon Instruments Europe, Netherlands). Image analysis was carried out in ImageJ (http://imagej.nih.gov/ij/). Pearsons coefficients were calculated using the JaCoP ImageJ plugin [57 (link)].

Transfected HEK-293 cells were cultured on 8-well glass chamber slides (Thermo Fisher Scientific) and transfected with pluc2-iRFP720, pluc2-iRFP670, and pTurboLuc using Fugene HD (Promega, Madison, MA, USA). After 24 h, HEK-293 cells were fixed using 4% formaldehyde. Rabbit polyclonal luc2 antibody (1:100) and anti-rabbit immunoglobulin G (IgG) Alexa Fluor 488 secondary antibody (1:200; Thermo Fisher Scientific) were used for immunofluorescent staining.

Rat L6 myoblast cells (ATCC CRL-1458) were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% (vol/vol) FBS (Invitrogen, Carlsbad, CA). Cells were cultured at 37°C in a 5% CO₂ humidified incubator in normoxia. Once suspended, 10⁴ cells were seeds into 8-well glass chamber slides (Thermo Fisher Scientific, Australia) and incubated overnight.
At 80% confluency cells were transfected using X-Treme Gene 9 (Roche, Germany), according to the manufactures’ guidelines with a 1:3 DNA:lipid ratio. To prepare lipoplexes, (at room temperature) 50 μL of serum-free DMEM was combined with 0.45 μL of lipid reagent, followed by 150 ng of fluorescently labelled DNA. The resulting mixutre was incubated for 15 min, and then added directly to cell preparations. During each experiment, 4 wells were transfected with each fragment and cells were imaged from all 4 wells. At all times cells were maintained in DMEM containing 10% FBS.
For cytoplasmic measurements, the cells were imaged between 4−8 hr after introduction of the lipoplexes, whereas, nuclear measurements were imaged after 36 hr. Cell nuclei were counterstained with Hoechst 33342 (1 μg/mL).