Lsr 1

BMDMs were detached with 10 mM EDTA/PBS solution. The pellets were collected by centrifugation at 200 g for 5 min and washed with cold staining buffer (PBS without Ca²⁺ and Mg²⁺ containing 0.5% BSA). BMDMs were resuspended in staining buffer at a final cell concentration of 1×10⁶/50 μl, incubated with 0.5 μg of purified anti-mouse CD16/CD32 Ab for blocking non-specific Fc-mediated interactions and then with anti-mouse CD284 (TLR4) PE (0.01 μg/μl) Ab or mouse IgG2a κ isotype control Ab for 45 min on ice, and analyzed by flow cytometry (Becton Dickinson LSR I).
Mitochondria-associated ROS levels were measured in BMDMs by staining cells with MitoSOX (20 (link), 21 (link)). Briefly, cells were incubated for 30 min at 37°C in a 10 μM MitoSOX™ Red (Invitrogen-Molecular Probes) solution prepared in 4:1 (v/v) DMEM/PBS. Cells were harvested with 500 μl of trypsin-EDTA solution, centrifuged at 2300 g for 5 min, and resuspended in 3 ml of 2% (v/v) FBS/PBS. Stained cells were determined with a Beckman Gallios and data analyzed with FlowJo10 analytical software (TreeStar). The mean fluorescence of the cell samples was then normalized to the unstained group.

One million NPCs were harvested to single cell suspension in 1mL PBS, then fixed by addition of 3 mL of 100% ethanol and stored at 4°C for at least two hours. NPC pellets were washed once with 5 mL PBS. After removal of PBS, cells were resuspended in 1 mL of propidium iodide (PI) staining solution (0.1% (v/v) Triton X-100, 10 μg/mL PI, and 100 μg/mL RNase A in 1X PBS). WS and TD NPC samples were analyzed by FACS on a Becton Dickinson LSRI, and gating of subG1 population (cells with fragmented DNA) was examined using FLOWJO-Flow Cytometry Analysis Software.