Celltirf

RhoA biosensor live-cell imaging was acquired with an oil objective lens (NA 1.4, Olympus, Tokyo, Japan) on a total internal reflection fluorescent (TIRF) microscope (CellTIRF, Olympus, Tokyo, Japan). Cells were plated on glass-bottom cell-culture dish before imaging. For RhoA biosensor imaging, the interval was 10 min. Cells were maintained in DMEM media supplemented with 10% FBS (PAN, Biotech, Aidenbach, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C throughout the imaging process. Images were acquired for 6 h. For cytokinesis live-cell imaging, MEFs were plated onto 30-mm cell-culture dish (Corning, Corning, NY, USA), and images were acquired using a 10× dry objective lens (NA 0.3, Olympus, Tokyo, Japan) on a deconvolution microscope (CellTIRF). Single-plane multipoint acquisitions were captured every 5 min within a 37 °C chamber.

Cells were fixed with 4% buffered paraformaldehyde, and permeabilized using 0.5% Triton X-100 for 15 min. The fixed cells were blocked with CAS-Block (Invitrogen, San Diego, CA) at 25 °C for 1 h. The cells were then incubated with primary anti-FAK, anti-vinculin, anti-Orai1 (Santa Cruz, Santa Cruz, CA), anti-STIM1 (abcam, Cambridge, UK), anti-paxillin (BD Biosciences, San Jose, CA), or anti-talin (Millipore, Billerica, MA) antibody overnight at 4 °C. In addition, the cells were stained with goat anti-mouse IgG conjugated with Alexa 488 or goat anti-rabbit IgG conjugated with Alexa 594 (Molecular Probes, Eugene, OR) for 1 h. The fluorescence images of the focal adhesions were acquired and analyzed using a total internal reflection fluorescence microscope (cell^TIRF; Olympus, Tokyo, Japan) with 491 nm laser. The FV10-ASW software was used to analyze focal adhesion proteins.

The detection system was set up on an epi-fluorescence microscope (Olympus IX81). Diode lasers (Toptica Photonics, Munich, Germany) were used for selective fluorescence excitation of GFP, YFP and Cy5/DiD at 488 nm, 514 nm and 640 nm, respectively. A 405 nm diode laser (Toptica Photonics, Munich, Germany) was used for bleaching of GFP/YFP fluorescence. Samples were illuminated in total internal reflection (TIR) configuration (CellTIRF, Olympus) using a 60 x oil immersion objective (NA  =  1.49, APON 60XO TIRF, Olympus, Munich, Germany). After appropriate filtering using standard filter sets, fluorescence was imaged onto a CCD camera (Orca-R2, Hamamatsu, Japan). Samples were mounted on an x-y-stage (CMR-STG-MHIX2-motorized table; Märzhäuser, Germany) and scanning of larger areas was supported by a laser-guided automated focus-hold system (ZDC-2; Olympus). For FRAP experiments single patterns were photobleached with a laser pulse (405 nm) applied for 100 ms. Recovery images were recorded at indicated time intervals. FRAP images were analyzed using the Multimeasure plugin of ImageJ [27] (link). Data were normalized by the pre-bleach image and curve fitting was done using Graphpad Prism. Resulting FRAP curves were plotted based on the standard error of the mean (SEM) and fitted using a bi-exponential equation. Kinetic FRAP parameters were directly obtained from curve fitting.