Acuri c6 plus

Phagocytosis in microglia cells was determined using the green fluorescent latex beads (Sigma, L1030-1ML), which were pre-opsonized in fetal bovine serum (FBS) (1:5 ratio) for 1 h at 37°C. Subsequently, the FBS with the beads was added to the wells to obtain a final concentration of beads = 0.1% (v/v) as previously reported in Lian et al. (2016) (link). After the cell treatment described previously, cells were incubated with the beads during 6 h at 37°C. We also incubated the experiment at 4°C for 6 h as a negative control.
The percentage of phagocytosis of the microglia was analyzed using a flow cytometer (BD Acuri C6 Plus). After the 6-h incubation ended, the culture medium containing the beads was discarded and the cells were washed twice with sterile 1 × PBS at 37°C. Cells were detached using 1 ml of the PBS/EDTA/EGTA/GLUCOSE solution (1 × /1 mM/1 mM/1 mg/ml) and resuspended in 100 μl of 1 × PBS for analysis in the BD Acuri C6 Plus flow cytometer in the channel FL1-A.

Cell apoptosis was determined by Annexin V, FITC conjugate kit (BD Pharmingen 556547). Cells were harvested and diluted 1 × 10⁶ cells/ml. The diluted cells were stained with Annexin V, FITC conjugate kit according to the manufacturer's protocol. The stained cells were analyzed by BD Acuri C6 Plus (USA San Jose, CA, USA).

Lipid accumulation was determined by Nile Red (Santa Cruz biotechnology sc-203747). Cells (3 × 10⁵ cells/well) were seeded in 6-well plate and treated with 0.5 mM FFA for 24 h. Cells were harvested and diluted 1 × 10⁶ cells/ml. The diluted cells were stained with 0.125 μg/ml of Nile Red for 10 min at a 37℃ incubator. The stained cells were analyzed by BD Acuri C6 Plus (USA San Jose, CA, USA).