Orca flash 4.0 digital

Cell viability was evaluated by LIVE/DEAD® Cell Imaging Kit (488/570) (R37601, Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol. Cells were labeled after the 18 h adhesion period and prior to removal of the seeding stencil. In order to evaluate the distribution of cells on the microwell array substrate, cell nuclei were labeled with the NucBlue® Live ReadyProbes® Reagent (R37605Thermo Scientific). The fixtures were disassembled and the stencil film removed as described, then the microwell array substrates were transferred to a six well plate containing fresh cell culture medium and the nuclear label. Cells were incubated for 10 min and then imaged via wide-field microscopy (Fig. 1i). The microwell array was imaged by phase contrast and the nuclear and viability stains were imaged via wide-field epi-fluorescence. The excitation and emission wavelengths in nm were: 360/460 to detect nuclei (blue), 485/540 to detect live cells (green), and 540/600 to detect dead and dying cells (red). All images were collected using an inverted, Nikon TE2000-U fluorescence microscope with a 4× plan apo lens, NA = 0.2, and a Hamamatsu Orca Flash 4.0 digital CMOS camera. The entire microarray was imaged in each channel by stitching individual image fields. Image acquisition was automated using NIS-Elements software.

Scrambling was assessed in single cells by elevating intracellular [Ca²⁺] via the patch pipet solution during patch clamp recording as described previously (Yu et al., 2015 (link)). Binding of Annexin-V-AlexaFluor-568 to patch-clamped cells during voltage-clamp recording was imaged with a wide-field Zeiss Axiovert 100 microscope using a 40X NA 0.6 LD-Acroplan objective. Images were acquired with an Orca-FLASH 4.0 digital CMOS camera (C11440, Hamamatsu) controlled by Metamorph 7.8 software (Molecular Devices). Annexin-V-AlexaFluor-568 was added to the normal extracellular solution before patch clamping the cell. After whole-cell recording was established with an intracellular solution containing 200 µM free Ca²⁺ the accumulation of Annexin-V on the plasma membrane was imaged at 1 min intervals synchronously with voltage clamp recording.

Imaging was performed using spinning-disc confocal microscopy with a 60 × 1.40 numerical aperture objective lens (Plan Apo λ, Nikon, Tokyo, Japan). A CSU-W1 confocal unit (Yokogawa Electric Corporation, Tokyo, Japan) with three lasers (488, 561, and 640 nm, Coherent, Santa Clara, CA) and an ORCA-Flash4.0 digital CMOS camera (Hamamatsu Photonics, Hamamatsu City, Japan) were attached to an ECLIPSE Ti-E inverted microscope (Nikon) with a perfect focus system. A stage-top incubator (Tokai Hit, Fujinomiya, Japan) was used to maintain the same conditions used for cell culture (37°C and 5% CO₂). For light illumination, a Mosaic-3 digital mirror device (Andor Technology, Belfast, UK) and a 488 nm laser (Coherent) were used. The microscope and attached devices were controlled using Metamorph (Molecular Devices, Sunnyvale, CA).