Csu x filter wheel

The cells were cultured at 37 °C and 5% CO₂ in a black 24-well glass-bottomed plate in Dulbecco's modified Eagle's medium containing nonessential amino acids (Life Technologies), 10% heat-inactivated fetal bovine serum (Life Technologies) and penicillin/streptomycin (100 units/ml, 100 mg/ml, respectively). Transfection of cells was carried out with 0.5 μg of DNA and 2.5 μl of Lipofectamine 2000 in 100 μl of OptiMem® for 16–48 h at 37 °C. The cells were imaged 16–48 h post-transfection in 2 or 1 ml of OptiMem®. The cells were imaged at 37 °C (OKO lab incubation chamber) with a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a 40×/NA1.3 oil immersion objective, and a 3i Laserstack as excitation light source (568 nm). Emitted light was recorded through a band bass filter (Yokogawa CSU-X filter wheel) by a CMOS at 1152 × 1656 pixel size. Time-lapse images were recorded at 2-s intervals for 3 min (acquisition time, 500 ms). Elliptical regions of interest were stacked, and fluorescence intensities were analyzed using ImageJ program. The data obtained from 13–30 cells was plotted and analyzed in OriginPro 9. Fluorescence decay times were determined from single exponential fits to the data.

HeLa cells stably expressing actin-GCaMP6s were plated on 35-mm glass-bottom dishes and challenged with 10 μM ionomycin or 500 μM histamine. Fluorescence over time was recorded on a 3i Marianas spinning-disk confocal microscope (3i, Denver, CO, USA) equipped with a Zeiss AxioObserver Z1, a ×40/1.3 Plan-Apochromat oil immersion objective, and a 3i Laserstack as excitation light source (488 nm for actin-GCaMP6s). Emitted light was collected through a single bandpass filter (CSU-X filter wheel; Yokogawa, Tokyo, Japan) onto a complementary metal-oxide semiconductor (CMOS) camera (Orca Flash 4.0; Hamamatsu, Hamamatsu, Japan). Images were collected at 1 frame every second (histamine) or every 5 s (ionomycin) using SlideBook v.6 software (3i) and processed on ImageJ. Data obtained from 10 cells was normalized and plotted on Prism 6 (GraphPad Software, La Jolla, CA, USA).

HeLa cells stably expressing actin-GCaMP6s were plated on 35-mm glass-bottom dishes and synchronized using thymidine-nocodazole protocol. Before the second release, cells were loaded with 2 μM diazo-2-acetoxymethyl ester (diazo-2-AM; a kind gift from Alexei Tepikin, University of Liverpool) for at least 30 min at 37°C.
Cells were then released and examined on a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a ×40/1.3 Plan-Apochromat oil immersion objective, and a 3i Laserstack as excitation light source (405 nm for diazo-2-AM photolysis; 488 nm for actin-GCaMP6s). Emitted light was collected through single bandpass filters (CSU-X filter wheel; Yokogawa) onto a CMOS camera (Orca Flash 4.0; Hamamatsu). Experiments were carried out at 37°C and 5% (v/v) CO₂ (incubation chamber from Okolab, Naples, Italy).
Photolysis of diazo-2-AM was performed by illumination with 405 nm laser light at 10% power for 10 ms for rapid chelation of Ca²⁺ at the centrosome or cytoplasm (elliptical region of interest) after 1–3 frames. Then, cell division was monitored by collecting images every minute using SlideBook v.6 software and processed on ImageJ and GraphPad Prism 6.