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Figure 2. The pbMEC and MAC-T properties in FBS-free medium. (A) Cell count of pbMEC from Day 4 to Day 12 after the isolation from fresh tissue, in six-well multiwell culture dishes. (B) The picture of cultured pbMEC shows the heterogeneous population of epithelial cells (red arrow) and fibroblast-like cells (white arrow). Scale bar: 100 μm. (C) Enrichment of epithelial cells over fibroblasts as means of mRNA expression of keratin 18 (KRT18)/vimentin (VIM) in FBS-containing medium with or without preplating and FBS-free medium. (D, E) mRNA expression in pbMECs of cell-type (D) and differentiation (E) markers on the isolation day (Day 0), at 50% and 80% confluency, and at 80% confluency at the first sub-passage (P1): keratin 18 (KRT18), keratin 14 (KRT14) for epithelial identity, vimentin (VIM) as fibroblastic marker, prolactin hormone receptor (PRLR), progesterone receptor (PR), casein alpha (CSN1S1) and casein kappa (CSN3) coding genes as differentiation markers. (F) Keratin 14 and 18 and vimentin mRNA expression for MAC-T cells cultured in FBS 10% or FBS-free medium. The mRNA expression data are depicted as 2^-ΔΔCt[47], values are mean ± SD of four independent replicates, and Kruskal-Wallis and Dunn’s multiple comparisons tests (C-E) or Wilcoxon test (F) were performed. The differences were considered significant when P < 0.05, (marked with “*” on the graph). pbMEC: Primary bovine mammary epithelial cells; FBS: fetal bovine serum.