Isolation of Decidual Leukocytes

Leukocytes from decidual tissues were processed, as previously described 28 (link). Briefly, decidual tissues were minced and digested with DNase I (150 µg/mL, D5025; Sigma-Aldrich, Saint Louis, MO, USA), collagenase (1 mg/mL, BS164; Biosharp, China), and hyaluronidase (1 mg/mL, H3506; Sigma-Aldrich, St. Louis, MO, USA) in the Roswell Park Memorial Institute-1640 culture medium. Ten milliliters of this enzyme cocktail was used per 1 g wet weight of tissue, with pulsed digestion of 3 × 20 min at 37 °C with stirring. After each incubation, the tissue was allowed to settle and the supernatant containing the released cells was removed. Finally, the dispersed cells were filtered through a metal sieve and silk, and washed twice with phosphate-buffered saline (PBS, Biosharp, China). The suspensions were loaded onto a Percoll (BS909; Biosharp, China) density gradient to purify the leukocytes. Decidual immune cells with densities of 25-50% were collected.