Sunbright oe 040cs

To monitor and localize ROS production by live confocal imaging, mast cells (1.5 × 10⁶ cells) were immobilized overnight in MatTek glass bottom microwell dishes (MatTek Co., Ashland, MA) using a biocompatible anchor for membrane (BAM; SUNBRIGHT® OE-040CS, NOF Corporation, Tokyo, Japan). To label granules and detect ROS, cells were incubated with a cocktail of probes (50 nM LysoTracker^TM Red DND-99 and 5 μM CellROX^TM Deep Red) for 30 min. Cells were subsequently washed and kept in PBS. Images were immediately recorded at the indicated time intervals at 37 °C and 5% CO₂ at baseline (initial 72 min), and after addition of mefloquine (for another 112 min) using a Nikon Ti2-E microscope, equipped with an X-LIGHT V2 L-FOV spinning disk with a pinhole size of 60 µm (Crest Optics). A 100X/1.45 NA oil objective (Nikon) and a Prime 95B 25 mm camera (Photometrics) were used to capture the images. To monitor cell death by live confocal imaging, BAM-anchored WT and serglycin^−/− mast cells (1.5 × 10⁶ cells) were incubated with a cocktail of Annexin V and DRAQ7 in a glass bottom 6-well plate (MatTek Co.). Thereafter, cells were treated with either PBS or mefloquine and images were immediately recorded at indicated time intervals, at 37 °C and 5% CO₂, through a 40X/0.6 NA air objective on a Nikon Ti2-E microscope.

Cells were immobilized either on petri dishes (Nunc 263991, ThermoFisher Scientific) or LigandTracer MultiDishes 2x2 (Ridgeview Instruments) for real-time binding assays, essentially as previously published (42 (link)). In brief, a biomolecular anchor molecule (BAM) (SUNBRIGHT^® OE-040CS, NOF Corporation) was dissolved to 2 mg/ml in ddH₂O water and circular drops of 400 µl were carefully placed onto the dishes and incubated for 1 h at room temperature. After carefully aspirating the BAM solution, cells suspended in PBS (due to differences in size 7.5*10⁶ cells/ml for Ramos, Daudi, P493.6 and LCL1.11, 2.5*10⁶ cells/ml for K562) were placed onto the BAM coated spots. Human primary B cells were resuspended in RPMI 1640 (supplemented with 1 mM sodium pyruvate, 100 µg/ml penicillin-streptomycin, 2 mM L-glutamine and 100 µM MEM non-essential amino acids) and 2*10⁶ cells and seeded on BAM coated spots. Cells were then incubated for 40 min at room temperature. Cells that did not attach were carefully removed and cell culture medium was added. Seeded cells were kept a humidified incubator at 37°C with 5% CO₂ and used for experiments the next day.