Amaxa nucleofector kit

Plasmids encoding SNAP-CLC were electroporated into dissected neuronal suspensions using an Amaxa Nucleofector Kit (Lonza) at DIV0 before plating on 35-mm gridded, glass-bottom MatTek dishes (part no. P35G-1.5-14-CGRD). At DIV14, neurons were stained with 0.5 μM Janelia Fluor 549 at 37 °C for 1 h, followed by incubation in original culture medium at 37 °C for 2 h before fixation for immunofluorescence or CLEM. Labeled neurons were imaged and their coordinates on the MatTek dishes recorded using fluorescence microscopy and bright-field differential interference contrast microscopy, respectively. Then neurons were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed in 0.1 M sodium cacodylate buffer containing 1% OsO₄ and 1.5% K₄Fe(CN)₆ (Sigma-Aldrich), en bloc stained with 2% aqueous uranyl acetate, dehydrated, and embedded in Embed 812. The nerve terminals expressing SNAP-CLC were relocated (based on the prerecorded coordinates), sectioned, and imaged. Ultrathin sections (60 to 80 nm) were observed with a Philips CM10 microscope at 80 kV, and images were obtained with the iTEM soft imaging system and a Morada 1k × 1k CCD camera (Olympus). Except when noted otherwise, all reagents for EM were obtained from EMS.

Tet-Plk4–expressing HUVECs were electroporated with a dual-chain Rac1 FRET biosensor (gift of Klaus Hahn, University of North Carolina at Chapel Hill; Kraynov et al., 2000 (link)) and centrin::tdTomato, using an Amaxa Nucleofector Kit (VPB-1492; Lonza) and then plated on fibronectin-coated (5 μg/ml) glass-bottom dishes. ECs were fixed (4% PFA) after overnight incubation with DOX to induce centrosome overamplification. For FRET imaging, a 453-nm laser was used for excitation, and both CFP (470/24 nm) and FRET/yellow fluorescent protein (535/40 nm) emission channels were collected with an Olympus 60×/NA 1.4 objective on an Olympus FV1200 confocal microscope. All images were acquired using the same parameters, optimized to reduce photobleaching. FRET images were processed as described previously (Kardash et al., 2011 (link)). Briefly, the acquired CFP/FRET ratios were corrected for background in ImageJ. Single-cell images were segmented in each channel in ImageJ using the minimum and mean threshold method, and regions of interest were analyzed. FRET ratio images were produced by dividing the processed FRET image by the CFP image. Individual cell mean FRET/CFP values corresponding to Rac1 activation were obtained by calculating the mean pixel value of the FRET ratio image for each single cell.