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rmKlotho was added at each culture medium replacement.
DMXAA was added by culture medium replacement 12 hr later.
For the latter, the manual culture medium replacement process in the conventional static cell culture practices normally leads to a fluctuating culture environment.
For experiments with both cDNA and siRNA transfections, the cDNA transfection was performed 1 day after the siRNA transfection with culture medium replacement, and the cells were collect 2 days after the cDNA transfection.
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In addition, only a few floating cells (5% in ESCs) were seen in the first 24 hours of culture, and very few floating cells were observed in the following cultures after medium replacement at 24 hours timepoint, indicating that decrease of labeled cells is not due to the selective death of these cells either.
The two main areas of focus were cell culture medium composition and replacement frequency relative to a base case of incubating constructs in medium supplemented with just serum and replaced weekly.
An explanation for that could be the daily changing of culture medium allowing the replacement of nutrients and discard of planktonic cells, which favors the growth of sessile bacteria.
The following day, culture medium was replaced with 1 μg/mL doxycycline (Dox; LKT Laboratories) in 20% KSR replacement media.
The culture medium was replaced every 72 h.
The culture medium was replaced daily.
Twenty-four hours after medium replacement, culture medium was harvested as conditioned medium (CM) from CNE2 cells (CNE2-CM), and used for the incubation of PBLs.
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