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Cells were grown in petri dishes (diameter 55 mm) until the total cell number reached 5×105.
The media change step was repeated until the total cell number exceeded 5 × 10 cells/ml.
In order to meet the requirement of Affymetrix gene microarray hybridization (i.e. at least 1 μg total RNA can be extracted), all the cell lines were cultured at least for three generations until the total cell amount was more than 10.
To determine differences in self-renewal capacity of cells from SP and non-SP fractions, 10 000 cells were isolated from each fraction and cultured for 2 3 weeks until the total cell number was increased to approximately one million cells, a number required for adequate SP analysis.
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The lysates were sonicated for 10 sec×2 and centrifuged at 10,000 g for 10 min; the supernatant contained the total cell extract and was stored at −20°C until used.
Likely reflecting progressive cellular accumulation, the total cell numbers of epithelial cells [21], alveolar macrophages [20], and migratory CD11b cells continue to increase until 14 days after pneumonectomy.
Lane2, the total cell lysate.
The supernatant contained the total cell lysate.
The small cell size can be expanded until either the total small cell load or the load in ABS exceeds the nominal load, i.e. CRE adaptation needs to take into account both load limits.
The supernatant containing total cell lysates were collected and stored at −80°C until use.
Mutant cells continued to accrue until the total population size reached 10 cells (27 generations), the number of parent cells plated onto selective medium.
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