Eclipse ti microscope

To detect intracellular ROS generation, a 2′,7′-dichlorofluorescin diacetate fluorescent probe (DCFH-DA, S0033S, Beyotime) was applied according to manufacturer’s instructions. In brief, chondrocytes were seeded on 12-well plate (1×10⁶/well), pretreated with or without TubA, and subjected to TBHP for 6 hours. After drug administration, the medium was replaced with DMEM/F12 containing 15 μM DCFH-DA for 30 minutes at the incubator with 37° C and 5% CO₂. Sequentially, chondrocytes were washed with PBS three times for removing unwanted DCFH-DA and immediately put on the worktable of the Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan) for image capture. The fluorescence intensity was analysed by ImageJ software (version1.52p). The mitochondrial membrane potential assay was evaluated by the Mitotracker Green fluorescent probe (C1048, Beyotime) according to the manufacturer’s instructions. Chondrocytes were incubated as above, treated with 100 nM Mitotracker Green for 30minutes at 37° C and then labelled with DAPI for 5 minutes. The images were also visualized by the Nikon ECLIPSE Ti microscope, and the intensity was measured by ImageJ software.

Cells were plated onto 35 mm-glass bottomed dishes (Greiner Bio-One, Monroe, NC, USA) and incubated on the microscope stage at 37 °C in humidified 5% CO₂. For monitoring membrane permeabilisation, cells were incubated with PI (5 μg/ml). A Zeiss LSM710 (Oberkochen, Alemania) confocal microscope with a Plan-apochromat × 63 1.3 NA oil immersion and × 40 1.3 NA objectives was used to visualise release of IL-1β. Image capture was performed using the ‘Zen 2010b SP1' Zeiss software. Alternatively, macrophages were imaged with a Nikon Eclipse Ti microscope equipped with a 40 × /0.60S Plan Fluor objective and a digital Sight DS-QiMc camera (Nikon, Tokyo, Japan) and the NIS-Elements AR software (Nikon). Time-lapse microscopy images were quantified either with ImageJ (US National Institutes of Health, Bethesda, MD, USA) or Cell Tracker (version 0.6, Pittsburgh, PA, USA).^{42 (link)} To quantify changes in plasma membrane dynamics over the time, macrophages were labelled with CTB-AF647 (1 : 1000 dilution) for 30 min at 37 °C and imaged using the Nikon Eclipse Ti microscope as stated above. Inverted fluorescence images converted to grey scale were used for quantification of the mean grey value as relative fluorescence units (RFUs) in different regions of interest of the plasma membrane of each cell using ImageJ (US National Institutes of Health).

Time-lapse image datasets of individual mother cells trapped in microfluidic devices were obtained from the Murat Acar and Peter Swain lab. The datasets were used to compare the performance of our cell division tracking pipeline, as described in the main text. Data from the Acar lab were generated on a Nikon Ti Eclipse using ×40 brightfield imaging with a 10min-interval and a single z-stack, as previously described (Liu et al., 2015 (link)). Data from the Swain lab were obtained using a Nikon Ti Eclipse microscope using ×60 brightfield imaging, a 2.5min-interval (Crane et al., 2014 (link); Granados et al., 2018 (link)), and 5 z-stacks combined into a single RGB image and used as input to the classifier. We also used a separate trap design from our own lab that is similar to a previously reported design (Jo et al., 2015 (link)) which was imaged on a Nikon Ti Eclipse microscope using a ×60 phase-contrast objective.