Fv3000rs

Lewis cells were seeded onto 24-well plate or confocal dish with a density of 2 × 10⁵ cells, then incubated with DHA, MIL-101 NRs and ICG-DHA@MIL-101 NRs for 12 h. The cells were harvested for flow cytometry (flow cytometry) analysis (Cytoflex, Beckman Coulter, USA). The cells on confocal dish were fixed with paraformaldehyde, nuclear stained with Hoechst 33,342, then imaged using a laser scanning confocal microscopy (FV3000RS, Olympus, Japan).

H9c2 cardiomyocytes were donated by Dr. He from Hubei university of medicine [17 (link)]. Cells were cultured in high glucose dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS (Gibco, C11995500), 100 IU/mL of penicillin and 100 μg/mL of streptomycin, and incubated in 95% air, 5% CO₂. The detection of reactive oxygen species (ROS) and ∆Ψm in H9c2 cells was carried out according to the specifications of ROS detection kit (Beyotime, S0033) and mitochondrial membrane potential detection kit (Solarbio, CA1310). For the determination of ROS, 1 × 10⁵/well H9c2 cells were first cultured under normal conditions (control group), O₂ 1% or O₂ 1% + QLQX, and then incubated with reactive oxygen species sensitive dye 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution at 37 °C for 20 min. In order to measure ∆Ψm, cells were treated as described above and then incubated with 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) staining solution (5 mg/ml) at 37 °C for 20 min. After staining, cells were washed twice with JC-1 staining buffer and detected by fluorescence microscope (Olympus FV3000RS). Fluorescence is measured at excitation/emission 485/580 nm (red) and then at excitation/emission 485/530 nm (green). The results were analyzed with Image-Pro Plus software.

Mice were imaged on an Olympus FV3000RS laser scanning confocal microscope at 30 fps using a 60X/NA1.30 silicone oil objective with 1X optical zoom using the resonance scanner. This allows for simultaneous excitation and detection of up to four wavelengths. Ten 1-minute fields of view were analyzed per mouse using FIJI/ImageJ software. Background noise was removed using a median filter (1 pixel) prior to image analysis. Vascular area was measured in each field using the selection brush in the FITC (dextran) channel. Firmly adherent neutrophils were defined as neutrophils moving less than 1 cell body over the course of 1 minute (S1 Fig; S2 Video). The number of neutrophils per mm² vessel area was then determined for each animal. To estimate the blood flow velocity within the sinusoids, red blood cells (RBC) were identified using negative staining within the dextran channel [23 (link)] and their velocities were measured using MTrackJ plugin in FIJI/ImageJ [24 (link)]. Tracks were created by manually selecting the center of RBC through a series of frames. An average of 10 tracks in different vessel segments were created per field of view (S2 Fig). The mean velocities were calculated in the MTrackJ plugin using a frame interval of 33.3 ms and then analyzed to compare the control- and rhRod-treated groups.

Slides were fixed using 2% paraformaldehyde (Precision Technologies Pte Ltd, 15710) for 20 min, followed by neutralisation in 50 mM of NH₄Cl for 5 min. NDFs were then permeabilised in 1% Triton™ X‐100 (Millipore, 648462) and 0.1% SDS (Promega, V6551) for 5 min, and blocked in 0.2% gelatin in dPBS (Hyclone, Cytiva) for 30 min. Cells were stained with the indicated primary antibodies overnight at 4°C before staining with secondary antibodies at room temperature in the dark. These were all performed with intermittent PBS washing steps. An IX‐83 inverted widefield fluorescent microscope (Olympus) was used for the majority of the experiments except for Figure S1—supplement 8, which utilised Zeiss Axio Observer 7 HCS (Zeiss) and the confocal images in Figures 1b and 2a, which used Olympus FV3000RS inverted. Images were subsequently processed and quantified using ImageJ (Schindelin et al., 2012 (link)) using macros derived from John Lim's macro (A*STAR Microscopy Platform [AMP], A*STAR).

Cells were seeded into six-well plates. The next day, cells underwent different treatments for 24 h followed by fluorescence staining according to standard methods as described previously. Immunofluorescence images were visualized and photographed using a laser scanning confocal microscope (FV3000RS; Olympus, Tokyo, Japan). ImageJ (National Institutes of Health) was used to measure the mean fluorescence intensity (23 (link)) (Supplementary Method S1.5).