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0.5 5.0 ml of scratched with sterile dH2O fungal spores and hyphae were inoculated per flask.
The cells were seeded in T25 flasks with 52,000 cells per flask (~2100 cells/cm2).
Three cuttings per flask in a single desiccator where used.
Each treatment was done in triplicate including a minimum of 50 polyps per flask.
Stem cells were sorted (see below) only when their number was at least 1,000,000 cells per flask.
The amount of sonicate added per flask of stimulated cells originated from one flask of RTHDF fibroblasts.
This ensured minimal variation in elapsed lifetimes between the pollinators, but the numbers of foundresses per flask necessarily varied.
αB crystallin expression was induced with 0.25 mL of 1 M isopropyl-β-d-thiogalactopyranoside (IPTG) per flask.
Cells were transfected with expression vectors pcDNA3 or pcDNA3::Sim1, 6 µg of plasmid DNA per flask.
Thus, cells were split into 50-ml cell culture flasks (1×106 cells per flask) and transfected with 4 µg of plasmid DNA/flask.
Approximately 40% of planulae in the 20, 24 and 30°C treatments settled and metamorphosed into polyps (H = 5.57, p>0.05, around 50 fully developed polyps per flask).
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