Mouse J774A.1 (ATCC TIB-67) and human THP-1 (ATCC TIB-202) cells were purchased from American Type Culture Collection to model mouse macrophages and human monocytes, respectively. Leukocytes were cultured in Dulbecco’s modified Eagle’s medium (high glucose, Gibco, Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 0.1% L-ascorbic acid 2-phosphate sesquimagnesium salt (AA2P, Sigma) at 37 °C in a humidified 5% CO2 incubator.
In order to isolate cell membranes, cells were harvested using a cell scraper at 80–90% confluency and resuspended at a concentration of 2.5 × 107 cell mL−1 in ice-cold tris-magnesium buffer (TM buffer, pH 7.4, 0.01m tris and 0.001 m MgCl2) and 1 EDTA-free mini protease inhibitor tablet (Pierce, ThermoFisher Scientific) per 10 mL of suspension. Cells were denucleated using a sonicator (Vibra cell model 375, Sonics and Material, Inc., Danbury, CT) for 4 × 10 s, while cooling in an ice/water bath. The homogenate was mixed with 1 m sucrose to a final concentration of 0.25 m sucrose, and centrifuged at 2000 g and 4 °C for 10 min. The supernatant was collected and further centrifuged at 20 000 g and 4 °C for 30 min in order to collect leukocyte membranes. Membranes were washed with ice-cold TM-buffer with 0.25 m of sucrose and collected by centrifugation at 20 000 g and 4 °C for 30 min and stored at −20 °C for further use.
In order to isolate cell membranes, cells were harvested using a cell scraper at 80–90% confluency and resuspended at a concentration of 2.5 × 107 cell mL−1 in ice-cold tris-magnesium buffer (TM buffer, pH 7.4, 0.01