Dii labeled ox ldl

In a previous study the higher lipid content was shown to coincide with increased cell surface expression of CD36 on classical monocytes [21 (link)]. Thus, since CD36 is involved in oxidized low-density lipoprotein (ox-LDL) uptake, we used the CD14 beads isolated monocytes (Miltenyi Biotec, Bergish Gladbach, Germany) to assess differences in ox-LDL uptake and the upregulation of activation markers with and without FSH stimulation. Ox-LDL uptake was measured by use of DiI labeled ox-LDL (Thermofisher). Media of FSH stimulated or unstimulated monocytes were replaced by fresh medium and 5ug/ml DiI labeled ox-LDL was added for 3 hours allowing uptake [22 (link)]. Hereafter, the cells were washed with PBS and lifted with TripLE (Thermofisher) for flow cytometry. Ox-LDL uptake was measured by flow cytometry by measuring fluorescence in the PE Channel (Beckman Coulter CytoFLEX) and analyzed by use of FlowJo software (version 10.7.0.). Debris and doublets were excluded using forward scatter (FSC) and side scatter (SSC). Ox-LDL uptake was presented as a percentage of ox-LDL positive cells, and median fluorescence intensity (MFI) was used to measure the amount of ox-LDL uptake per cell.

Mouse peritoneal macrophages from female C57BL/6 mice were elicited by intraperitoneal injection of 2 mL of 4% thioglycollate medium (Sigma) 3 days before collecting. The cells were incubated in 24-well plates at 5 × 10⁵ cells/well in RPMI 1640 complete medium in a humidified atmosphere of 5% CO₂/95% air at 37 °C. After 3 h, non-adherent cells were washed away three times with PBS. The adhered peritoneal macrophages were incubated in medium for 24 h before use. To evaluate the effect of nalmefene on oxLDL uptake, peritoneal macrophages were treated with nalmefene (300 μg/mL) for 24 h after serum starvation for 3 h and then 5 μg/mL DiI-labeled oxLDL (Thermo Fisher Scientific) was added to the culture medium for 4 h.
After three washes with PBS, the cells were fixed in 4% paraformaldehyde phosphate buffer solution and mounted with Vectashield containing the nuclear dye DAPI (Vector Laboratories). The area of DiI-labeled peritoneal macrophages was measured, and the number of peritoneal cells was counted. oxLDL uptake by peritoneal macrophages was evaluated by the DiI-positive area per cell in 15 random microscopic fields from three independent wells in each experiment. The experiment was repeated seven times, and the mean areas from separate experiments were analyzed.