The cells were seeded at 2500 cells/cm in 24-well plates with the neurogenic medium (Thermo Scientific) (two replicates) or under growth medium (two replicates) and maintained for 6 days, changing the media every 3 days.
For chondrogenic differentiation in monolayer cultures (n = 5), 10 cells/cm were seeded in 24-well plates with the chondrogenic media described by Jäger et al. [ 53] (two replicates) or with growth medium (two replicates).
Cells from the 6 sheep were plated at 2 x 10 cells/cm in 24-well plates and cultured under osteogenic conditions (two replicates) or with growth medium (two replicates) for 21 days as previously described [ 39].
In this heterotrophic cultivation using various organic or inorganic compounds as a carbon source, the cultures were cultivated in 250 ml flasks containing 100 ml of each tested medium, with three replicates.
Control wells were filled with culture medium only (six replicates), while treatments contained 1, 10 or 100 nmol l−1 of rodent leptin (Shenandoah Biotechnology) or kisspeptin-10 (AnaSpec) with four replicates per treatment.
To test for frequency-dependent selection within microcosms, we isolated two genotypes forming different colony morphologies from each microcosm after 1000 generations of selection and competed them against one another at initial frequencies of 1∶10 and 10∶1 over four (block 1) and three (block 2) days of daily transfers into fresh medium, with two replicates at each frequency.
Briefly, 10,000 cell/well were seeded in semi-solid agar mixed with medium in six replicates in six well plates.
Human BM-MSCs that had just been thawed were seeded at a density of 2 × 10 cells/well in 6-well plates with the medium in five replicates.
We cut 5.5 mm mycelial plugs and transferred each into a flask with 30 ml of one of four different liquid media, ¼-mPmT, ¼-mPmT + glucose, ¼-mPmT + PGA, and ¼-mPmT + RGI, using three replicates per medium.
For 2-deoxyglucose titration, a range of 0.005 – 5.0 g/L 2-deoxyglucose and 5 mM cAMP (Sigma-Aldrich, St . Louis MO) were added as indicated to T. saccharolyticum microwell cultures supplemented with 5 g/L glucose or cellobiose, inoculated with 10% v/v of an exponentially growing culture in the same medium (one of three replicate experiments is presented in Figure 1).
