Round bottom facs tube

Neutrophil ROS production was measured through the oxidization of non-fluorescent dihydrorhodamine 123 to fluorescent rhodamine 123 by ROS using the Neutrophil/Monocyte Respiratory Burst Assay Kit (Catalogue number 601130, Cayman Chemicals, Ann Arbor, MI) according to manufacturer’s instructions [24 (link)]. In brief, 100 μl EDTA–anticoagulated whole blood and 10 μl of 5 μg/ml dihydrorhodamine 123 working solution were added to each 5-ml round-bottom FACS tube (Catalogue number 352054, Corning), mixed well, and incubated in a 37°C water bath for 15 min. To stimulate ROS production, phorbol 12-myristate 13-acetate (PMA; Catalogue number 400145, Cayman Chemicals) in dimethyl sulfoxide (DMSO) was added to each cell suspension to reach a final concentration of 200 nM or as indicated in the dose-response assay, and the same volume of PBS was added to controls. After incubation in a 37° C water bath for 45 min, 3 ml PBS was added to each tube followed by centrifugation of the cells (700 × g for 5 minutes at room temperature). Cell pellets were resuspended in 200 μl PBS for immunostaining (CD66abce-PE) and live/dead detection via flow cytometry as described above for neutrophil phagocytosis. ROS production was measured by detection of rhodamine 123 signal in the FITC channel of live CD66abce⁺ cells.

The respective expressions of pCRY2-mCherry-linker-RGS2box constructs and pNtermRGS2-GFP-CIBN [P5] were assessed by flow cytometry. Cells were seeded onto a poly-D-lysine (PDL)-treated 6-well plate 24 hours prior to co-transfection. Cells were trypsinized 24 hours post-transfection and washed three times with phosphate-buffered saline (PBS) (ThermoFisher, 14190250). Cells were re-suspended in flow buffer containing 1X Ca^2+/Mg²⁺-free PBS, 5 mM EDTA, 25 mM HEPES pH 7.0, and 1% FBS. Cells were subsequently strained with a 70 μm cell strainer (Sigma, Z742103) and collected in a 5 mL polystyrene round-bottom FACS tube (Corning, 352054). The flow analyses were performed on 4-laser BD LSRII Cell Analyzer (mCherry detection: green laser with 610/20 emission filter; GFP detection: blue laser with 515/20 emission filter), at a rate of approximately 4000 events/s, with 100,000 events collected for each condition. Three gates were applied to select for live single cells with expression of both cryptochrome components.