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On the third generation, L1 individuals were seeded in two 305 mM NaCl NGM-lite plates.

Frozen samples with >10 individuals of the A00, T00, D00 and M00 populations were thawed and after one passage, 10 individuals were seeded for each of rR1-# replicate populations; where r designates the salt regime (Sudden, Gradual or Control), R the reproduction system (Androdioecy, Trioecy, Dioecy, Monoecy), and # the replicate population number.

In this case, a population of 500 individuals was seeded.

Generally, for each experimental condition 48 individual wells were seeded with randomly selected cellular aggregates and experiments were repeated three times.

For foci formation, early-passage MEFs (P0-P1) derived from individual embryos were seeded (106 cells) in plates of 10 cm diameter and cultured overnight in complete medium containing 10% FBS.

Responders from individual mice were seeded at 106 cells/well for total splenocytes and stimulated with medium or 10 µg/ml of each peptides from a library of murine overlapping 30-mer PrP peptides (Table 1).

Supernatants were mixed and individual cells were seeded at 50 100 cells/ml in culture flasks in proliferation medium (Fig. 3A1, plating progenitors).

For every concentration step, 4 individual wells were seeded and analyzed (n = 4) and experiments were repeated at 3-4 separate occasions (N = 3-4).

To generate ESD-EpiSCs (ESC-derived epiblast stem cells), ESCs or cell aggregates were dissociated into single cells after treatment with 0.05% Trypsin-EDTA at 37°C for 2 min. Individual cells were seeded at a density of 2.0 × 10 cells per 35-mm dish in CDM/AF.

Individual cells were seeded at 200 cells/mm on parylene-C and silicon oxide substrates that had been incubated for 2 h in Fn (50 μg/mL), albumin (5000 μg/mL) or Fn/albumin (50 and 5000 μg/mL) in PBS, at room temperature (20 °C).

To determine the Differential Stress Resistance to hydrogen peroxide of serum from CR and WD individuals, human fibroblasts were seeded in 96-well plates at a concentration of 3000 cells/well in 86% D-MEM low glucose w/o phenol red supplemented with 1% Sodium Pyruvate, 1% non-essential amino acids, 1% GlutaMAX and 1% antibiotics (GIBCO, Carlsbad, CA, USA).