*4.5. Flow Cytometry*

Implantation sites were separated from myometrium and tissue finely minced in 100 uL of ice cold PBS. The finely minced tissue was digested in RPMI 1640 (Gibco) containing 1 mg/mL Collagenase A (Roche) and 0.1 mg/mL DNase I (Roche) for 30 min at 37 ◦C with horizontal shaking at 250 rpm. Mechanical dissociation by repeated passage through an 18 G needle was performed twice at 15 min intervals during enzymatic digestion. Ice cold RPMI containing 3% FBS (Atlanta Biologicals, R&D

Systems, Minneapolis, MN, USA; RPMI-3)) was added and cells were filtered through a 100 µM nylon cell strainer.

Cells were stained in RPMI-3 for 30 min on ice in the dark with the following fluorochrome-conjugated anti-mouse antibodies: APC/Fire™ 750 anti-CD31 (BioLegend, clone MEC13.3, San Diego, CA, USA) and Super Bright 600 anti-CD45 (eBioscience, clone 30-F11, Carlsbad, CA, USA). Cells were washed twice and then resuspended in RPMI-3 containing 1µg/mL DAPI ((ThermoFisher, Carlsbad, CA, USA) and 2 mM EDTA (Corning, Carlsbad, CA, USA). Flow cytometry was performed using an Attune NxT (ThermoFisher, Carlsbad, CA, USA), LSRII (Becton, Dickinson and Company Sparks, MD, USA), or Fortessa (Becton, Dickinson and Company Sparks, MD, USA) flow cytometer, and analysis was performed using FlowJo™ for Mac v10.6.1 software (Becton, Dickinson and Company Sparks, MD, USA). Live endothelial cells were identified as DAPI–CD45–CD31<sup>+</sup> cells.
