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Two flask of each type of cells were used every day to have precise average count of the cell.
After 12 days one flask of each culture was pelleted and re-suspended in an appropriate nitrogen free media and cultivated for a further 6 days.
The physicochemical parameters, including pH, redox potential (ORP), the concentration of dissolved ferrous iron, total iron, copper ion, zinc ion and sulfate ion in the solution were monitored and a flask of each experimental group was removed for DNA extraction on day 6, 12, 21 and 30 (Xiao et al. 2015).
The three cultures were diluted to an O.D. of 0.02 in LB and grown at 37°C to an O.D. of 0.1 where they were split and 20 µg/ml of 2-AP was added to one flask of each culture.
The first flask of each cell line constituted the experimental control.
As an additional negative control, one flask of each of the three cell lines was left untreated but monitored once a day alongside the TSV-injected flasks.
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Fourteen flasks of each line were seeded.
After heating in water bath at ±37°C for 30 min, 0.7 ml anticoagulated blood solution was injected into the flasks of each group, then shaken and heated at ±37°C for 60 min. The supernatant was removed after centrifugation for 15 min at 1,000 rpm.
To account for and eliminate biologic noise, RNA was isolated from three distinct flasks of each cell line.
Cells were collected from sub-confluent flasks of each experimental group (aSC, nSC and ASC before and after glial differentiation).
Six 2 l flasks of each strain were inoculated, with 2% (w/v) sorbitol as the sole carbon source in minimal medium (see above).
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