Ld magnetic bead columns

Implantation sites at E7.5 were dissected to separate the decidua from overlying myometrium, and the decidua was enzymatically disaggregated with Liberase 3 (Roche), DNase-I (Roche), and trypsin for 15 min on ice, followed by 45 min at 37°C with periodic trituration. EDTA was subsequently added to a final concentration of 5 mM, and cells were incubated for a further 15 min at 37°C. After digestion, DSCs were separated with LD magnetic bead columns (Miltenyi Biotec) according to the manufacturer’s protocol. Selection was carried out using microbead-coupled antibodies toward red blood cells (Ter-119; Miltenyi Biotec) and leukocytes (CD45; Miltenyi Biotec), together with antibodies against epithelial cells (CD326; University of Iowa hybridoma bank) and endothelial cells (CD102; Biolegend, clone 3C4), which were rendered magnetic though secondary application of microbead-coupled goat anti-rat IgG (Miltenyi Biotec). After isolation, DSCs from each biological isolate were split into two wells and cultured for 24 h at 0.5 × 10⁶ cells/well in 1 mL SFM4CHO medium supplemented with MEM nonessential amino acids, HEPES buffer, penicillin-streptomycin, L-glutamine, and sodium pyruvate, with or without 2 ng/mL TGF-β1 (InvivoGen).