500 mpl system

HAoECs were seeded into 12-wells plate and grown until confluence. Then, HAoECs were exposed to normoxia or IH for 6 hours, in presence or in absence of ML-7 (5 μM). After the stimulation, cells were washed once with PBS and incubated with dihydroethidium (3 μM, Sigma-Aldrich) for 30 minutes at 37 °C. Cells were detached with trypsin and suspended into 200 μl of PBS. Fluorescence was then determined by flow cytometry 500 MPL System (Beckman Coulter, Villepinte, France) with the MXP software (Beckman Coulter).

MP were stained with PKH26 dye (Sigma Aldrich) following the manufacturer’s protocol. Briefly, MP were labelled with 2 μM PKH26 dye in 0.9% NaCl solution for 2 min at room temperature. An equal volume of FBS was added to stop the staining reaction. The MP pellet was recovered by centrifuging at 14,000 g for 45 min, washing in 0.9% NaCl, and was finally resuspended in 0.9% NaCl. This labelling procedure was efficient as confirmed by flow cytometry (93.5 ± 3.4% of MP population PKH26⁺, n = 4 independent labelling experiments).
Post staining, 10 μg/mL PKH26-labeled MP^Hh+ were incubated with 3T3-L1 preadipocytes for the indicated times either at 37 °C or 4 °C. At the end of the incubation time, MP internalization in 3T3-L1 was evaluated either by flow cytometry on trypsinized 3T3-L1 cells resuspended in 250 μl PBS solution (500 MPL system, Beckman Coulter, Villepinte, France), or by confocal microscopy of PFA fixed and DAPI counterstained cells.