Tcs sp8 aobs

Spheroids with diameter of about 250 μm were incubated with a final nanoparticle concentration of 0.5 mg/mL in cell media (0.2 mL/well). After a 24 h incubation, the spheroids were washed with PBS and incubated with 1 μg/mL Hoechst 33,342 and 5 μg/mL CellMask for 5 min at 37 °C, 5% CO₂. The spheroids were then washed and used for imaging using a confocal microscope. Confocal acquisitions were performed on live cells using a Leica TCS SP8 AOBS inverted confocal equipped with an immersion oil objective (20×/1.40/HCX PL Apo, Leica). For the acquisitions, the confocal settings^{36 (link)}, were as follows: pinhole 1 airy unit, scan speed 400 Hz unidirectional, format 2048 × 2048 (pixel size 280 nm). To eliminate any possible crosstalk between channels, images were collected with a sequential scan using the following laser lines and mirror settings: 405(100%) 410–483 nm; 488(25%) 495–550 nm; 561(100%) nm. Images were processed to enhance brightness using the ImageJ software (https://rsb.info.nih.gov/ij).
For the avoidance of doubt, we decided not to use spheroids larger than 250 µm in diameter for microscopy studies (as opposed to the 400 µm spheroids used for flow cytometry) since the transmitted light decreases with spheroid size. The use of 250 µm spheroids turned out to be the best compromise between ease of handling and light transmission at the core of the sphere.

Images were collected on a Leica TCS SP8 AOBS upright confocal microscope through LAS X (v3.5.2.18963) using a 63X/1.40 HCS PL Apo objective and 1× confocal zoom. The confocal settings were as follows: pinhole 1 airy unit, scan speed 400 Hz bidirectional, format 1024 × 1024. The white‐light laser was used with FITC 488 nm, Cy3 555 nm, Texas Red 594 nm and Cy5 647 nm laser lines. Images were collected using hybrid and photon‐multiplying tube detectors with the following detection mirror settings: DAPI 410–520 nm, Cy3 565–636 nm and Cy5 657–744 nm for IL‐1β/Iba1 images; and DAPI 410–478 nm, FITC 498–584 nm and Texas Red 604–749 nm for ASC/Iba1 images. Images were collected sequentially to eliminate crosstalk between channels. For live imaging, OHSCs were LPS‐primed (1 µg/ml, 3 hr) and then medium was replaced with phenol‐red‐free, serum‐free culture medium containing Hoechst (2 µg/ml; ThermoFisher) and isolectin GS‐IB4–AlexaFluor 594 conjugate (from Griffonia simplicifolia; IB4; 5 µg/ml; ThermoFisher) and incubated for 2 hr at 37°. OHSCs were subsequently covered with 1·5 ml phenol‐red‐free, serum‐free medium to allow lens immersion, and then nigericin (10 µm) was spiked into the culture medium underneath the insert. The OHSCs were then placed into a confocal microscope chamber heated to 37° and imaged for 60–90 min.

Live zebrafish were mounted ventrally on coverslips in 1% low-melting-point agarose containing MS222 (for live samples) and imaged using a Leica TCS SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope using a 10× dry lens or 20× glycerol lens. The temperature in the chamber covering the microscope was maintained at 28°C. Movies were recorded at an interval time of 5.45 min or 3.75 min per frame and a total time of 60 min or 120 min for neutrophils and macrophages, respectively.