Las x premium software

Cells were grown on 4-, 8- or 18-well µ-slides (ibidi, Gräfelfing, Germany) to the corresponding stages, rinsed twice in PBS pH 7.4 and fixed in 4% paraformaldehyde in PBS for 15 min, washed 3x in PBS for 5 min, permeabilized in 0.5% Triton X-100/PBS for 15 min and washed 3x in PBS for 5 min, all at room temperature. Cells were pre-incubated in 5% normal donkey serum (NDS) in permeabilization solution for 1 h at room temperature and incubated with primary antibodies (Table 3) overnight at 4 °C. After 3 × 10 min washes in PBS, cells were incubated with Alexa Fluor 488 and 555 secondary antibodies (Table 4) in 2.5% NDS in permeabilization solution for 1–2 h, washed 3 × 10 min with TBS (50 mM Tris-HCl pH 7.5; 0.9% NaCl) and incubated with 0.5 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 15 min, all at room temperature. After 3 × 5 min washes in TBS, cells were mounted in mounting medium (ibidi) and imaged on a DMi8 widefield microscope (Leica Microsystems, Wetzlar, Germany). Images were acquired with a DFC9000 GT sCMOS camera using LAS X Premium software (Leica Microsystems) and processed with Adobe Photoshop (Adobe, San Jose, CA, USA) software.

Cells grown in 6-well plates were directly visualized with a DMi8 widefield microscope (Leica Microsystems). Phase-contrast and fluorescent images were acquired with a DFC9000 GT sCMOS camera using LAS X Premium software (Leica Microsystems). For Ca²⁺ imaging, cells were grown in 35 mm µ-dishes (ibidi) until the desired stage. Before imaging, cells were rinsed once with recording medium (128 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 45 mM sucrose, 10 mM glucose and 10 mM HEPES; pH 7.4) and incubated in this medium with 4 µg/mL of Fluo-8-AM for 30 min at 37 °C, as described by Carola et al. [31 (link)]. Cells were washed once with recording medium to remove residual Fluo-8, replenished with fresh recording medium and immediately imaged on a DMi8 widefield microscope equipped with an on-stage incubator (Pecon/Leica Microsystems).

Images were captured and analyzed using a Leica SP8 white light pulsed laser confocal microscope and Leica LAS X Premium software at the Cell Sciences Imaging Facility at Stanford. Channels were selected and the exposure times were adjusted to optimize the images. Imaris software (Bitplane Inc., Concord, MA) was used for image capture^{62 (link)}.