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First, the observational design of the study makes it difficult to draw strong conclusions toward the optimal confluence stent technique.
All experiments showed optimal confluence (i.e., > 70%) except one, which displayed a confluence of about 50 60%.
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It was not possible to observe the trend of proliferation at longer times because, after 120 h, cells treated with the optimal concentration showed a confluence so high that it prevented further growth due to contact inhibition.
Each culture plate was passaged after 2 days of growth which we have previously shown was sufficient to achieve >80% confluence and was optimal for maintaining the pluripotency of the ESCs [17].
Initial studies found lamellar keratinocytes to reach 70 - 80% confluence under conditions optimal for skin keratinocytes, in a mean of 9.6 days ± 3.09 (n = 3), however cells were large, and unable to attach and proliferate upon sub-culture.
All experiments were carried out once the cells had reached 80 90% confluence and at their optimal passage number.
All experiments were performed on confluent cultures except for those experiments using antibody visualization, subconfluent cells of approximately 70% confluence were used for optimal antibody coverage.
Cell lines were subconfluent, and each cell line has been optimized in previous titration experiments so that cell type-specific optimal conditions for the seeding rate and confluence were obtained for the real-time measurement in these experiments.
rBM-MSCs at passage 3 (P3) were used in the adipogenic differentiation experiments, in which 80 90% sub-confluence was found to be optimal for cell differentiation that lasted for 2 3 weeks.
Cells were harvested from exponential phase cultures (at 50 75% confluence) by trypsinisation, counted and plated at optimal seeding densities in 48-well plates to assure exponential growth during the experiments.
At ROC analysis, an APD/LAD ratio of 0.63 measured 1 cm above the confluence of the iliac veins was the optimal cut-off for differentiating controls from patients with abdominal obstruction; this value was associated with 74% sensitivity and 96% specificity.
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