Glass bottomed dishes

J774A.1 cells or THP-1 cells were transferred to glass-bottomed dishes (NEST Biotechnology, Wuxi, China) and incubated overnight to 10⁶ or 5 ×10⁵ cells/well. The cells were treated with CLO variants as described above for 1 h, washed three times with PBS, and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 5% BSA in PBST (PBS with 0.1% Tween 20) for 1 h. To examine membrane binding, the cells were incubated with DyLight® 650 Anti-6 × His tag antibody (1:200 dilution, Abcam) for 1 h at room temperature. To examine ASC speck formation, the cells were incubated with ASC-antibody (1:1000, Abclonal) for 1 h and then incubated with anti-Rabbit IgG antibody conjugated with Alexa Fluor 594 (1:200, Abcam) for 1 h. Cells were washed as above with PBST and incubated with DAPI (Sangon, Shanghai, China) for 15 min. The cells were observed with a confocal microscope (Carl Zeiss LSM710, Jena, Germany).

Rat liver slices were prepared from the liver of an 8 week old Sprague Dawley rat. The liver slices were cut into 800 μm thicknesses using a vibrating-blade microtome in PBS buffer. Slices were incubated with 20 μM SE1 in PBS bubbled with 95% O₂ and 5% CO₂ for 2 h at 37 °C. Slices were then washed three times with PBS and transferred to glass-bottomed dishes (NEST) and observed in a spectral confocal multiphoton microscope. The TPM images were obtained at about 300 μm depth.

Large-format agarose pads were imaged using a Zeiss Axio Observer Z1 and ZEN 2 (blue edition) software, with the ZEN Tiles and Positions and ZEN Autofocus Modules installed. Images were captured using a Zeiss Axiocam 503 with 3X analogue gain. Using a low magnification 10X objective, each sample was localized on the pad. Approximately 24 fields-of-view were selected with a 100X Phase Contrast Objective (1.4NA), before capturing images using bright-field and fluorescent imaging. Fluorescence was excited using a Colibri Green LED (555/30 nm) and filtered at 590–650 nm. Exposure times were maintained across imaging sessions for Brightfield Images, and for fluorescent images. A software autofocus regimen was used before each field-of-view was captured. In the case of low-density samples, fields-of-view were manually chosen and increased in number. Raw images were saved as CZI files prior to processing and data extraction.
Time-lapse imaging was performed on 1.5% agarose pads embedded with 7H9 OADC medium containing ATc (Sigma, 100 ng/ml) and kanamycin (Roche, 20 µg/ml), in glass-bottomed dishes (NEST Biotechnology). Cells were maintained in an incubated chamber at 37°C and imaged every 15 min with a software autofocus regimen implemented between each frame.