Chicken serum

PGCs were cultured in 500 µl of high calcium FAOT medium containing 1.8 mM CaCl₂ in fibronectin-coated wells (24-well plate) for 48 hr (Figure 3—figure supplement 2) (Whyte et al., 2015 (link)). Subsequently, PGCs were transferred into PGC fibroblast medium and then refreshed every 48 hr by removing and replacing with 300 µl of PGC fibroblast cell culture medium. Adherent fibroblast-like cells were observed within 72 hr. Cells were then refed every two days and split 1:4 every four days. PGC fibroblast cell cultures were expanded to 85–90% confluency in 24-well plates before using for transfection, infection or western blot analysis. PGC fibroblast cells were maintained in cell culture media (Knockout DMEM (10829018, Gibco) with 10% ES grade FBS (16141061, Invitrogen), 1% chicken serum (Biosera), 0.1% 100xNEAA (Gibco), 0.1% Pyruvate (11360070, Gibco), 0.1% 100xGlutamax (Gibco: 35050–038), 0.5 mg ml⁻¹ ovotransferin (C7786, Sigma)) and 1% penicillin-streptomycin at 37°C with 5% CO₂.

Avian PGC culture medium contained 1 × B-27 supplement, 2.0 mM GlutaMax, 1 × NEAA, 0.1 mM β-mercaptoethanol, 1 × nucleosides, 1.2 mM pyruvate, 0.2% ovalbumin (Sigma), 0.2% sodium heparin (Sigma) 5 μg/mL in avian DMEM, a custom basal medium (a modification of knockout DMEM [250 mosmol/L, 12.0 mM glucose, and CaCl-free; ThermoFisher Scientific]). The following growth factors were added before use: human Activin A, 25 ng/mL (Peprotech); human FGF2, 4 ng/mL (R&D Biosystems); 0.2% chicken serum (Biosera). All reagents were purchased from ThermoFisher Scientific unless otherwise specified.

150,000 PGCs were incubated in 500 µl of high calcium FAOT medium containing 1.8 mM CaCl₂ in fibronectin-coated or gelatin-coated wells in 24-well plates for 48 h. Subsequently, the FAOT medium was replaced with PGC fibroblast cell culture medium and then refreshed every 48 h by taking out 300 µl and adding back 300 µl of PGC fibroblast medium. Adherent fibroblast-like cells were visible with 48 h of culture in PGC fibroblast medium. Cell culture medium was refreshed every 48 h and cells were split 1:3 once they are reached 85-90% confluency in 24-well plates. PGC fibroblast-like cell culture medium contains 10% ES-grade foetal bovine serum (Life Technologies #16141061), 1% chicken serum (Biosera #CH-515), 0.1% 100x NEAA (Life Technologies #11140050), 0.1% sodium pyruvate (Life Technologies #11360070), 0.1% 100x GlutaMax (Life Technologies #35050-038), and 50 ng/ml ovotransferrin (Sigma-Aldrich C7786) in Knockout DMEM (Life Technologies #10829018). PGC fibroblast cultures were maintained at 37 °C in a 5% CO₂ atmosphere.