Pha from phaseolus vulgaris

LPS from Escherichia coli O111:B4, PHA from Phaseolus vulgaris, H-89 [protein kinase A (PKA) inhibitor], forskolin (adenylate cyclase activator), and PGE2 were obtained from Sigma-Aldrich. AS-605240 [phosphatidylinositol-3-phosphate kinase (PI3K) inhibitor], PF-04418948 (EP2 receptor antagonist), and L-161,962 (EP4 receptor antagonist) were purchased from Cayman Chemical. Ficoll-Hypaque was obtained from GE Healthcare. Recombinant human TGF-β1, recombinant human M-CSF, recombinant human IL-4, and recombinant human GM-CSF were obtained from Miltenyi Biotech.

Briefly, blood from a female animal (NN03) was collected in a sodium-heparin vacutainer (Becton–Dickinson) and used for short-term (72-h) lymphocyte cultures as previously described^{89 (link)}. We used phytohemagglutinin (PHA from Phaseolus vulgaris, 20 μg ml^–1, Sigma Aldrich) as the mitogen. Additionally, we established primary fibroblast cultures under sterile conditions using small pieces (0.5 mm²) of ovarian tissue from NN03. The fibroblasts were incubated in MEM alpha containing nucleosides and GlutaMax (Thermo Fisher), supplemented with 20% fetal bovine serum (Atlanta Biologicals) and antibiotic-antimycotic (Thermo Fisher) at 30 °C with 5% CO₂. Metaphase chromosomes were obtained from both lymphocyte and fibroblast cultures by arresting cells with demecolcine (KaryoMax, Thermo Fisher; final concentration, 0.1 μg ml^–1), followed by hypotonic treatment with Optimal Hypotonic Solution (Rainbow Scientific) and fixation in methanol/acetic acid (3/1). Metaphase spreads were prepared on precleaned wet glass slides at room temperature. Chromosomes were stained with 5% Giemsa (KaryoMax, Thermo Fisher) in GURR buffer (Gibco). At least 30 metaphase spreads were captured and analyzed for karyotyping using an Axioplan2 microscope (Zeiss) and Ikaros (MetaSystems) software.