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The legal problem was solved with the 2007 Act and the ECR was thereafter supplemented with previously missing incident cases.
To assess the effect of hyperglycaemia, VSMCs were plated in DMEM containing 5 mM D-glucose for 48 hours and thereafter supplemented with 25 mM D-glucose (Sigma, St-Louis MO), 30 mM L-glucose (Sigma) or 30 mM mannitol (Sigma) for 24 or 48 hours.
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The next year, he published, in which he described 935 genera of plants, and shortly thereafter he supplemented it with, with another sixty (sexaginta) genera.
Thereafter, CM supplemented with or without 5 μM MG132 was added for further 34 h, and MTT assay was performed.
Older children, at least 10 years of age at the start of treatment, were interviewed directly, supplemented thereafter with caregiver interviews.
Blasticidin-resistant transfectants were sorted by fluorescence activated cell sorting (FACS) for mild EGFP expression and cultured thereafter in complete medium supplemented with 10 µg/ml of blasticidin.
Adherent cells were thereafter grown in DMEM supplemented with 10% serum.
And, finally, patients were kept in a non-fasting state at all times - glucose was administered on the first day, and thereafter balanced enteral nutrition, supplemented where needed by parenteral nutrition, was provided during the entire stay in the ICU.
Protoplasts were thereafter transferred to BCDAT medium supplemented with 40 μg mL-1 of G418 and cultured for 10 days.
Medium was thereafter changed to RPMI (Mediatech) supplemented with 0.5% pen/strep, 0.5% glutamate and 100 ng/ml activin A (R&D) for one day.
All the individuals used in this experiment belonged to a captive colony (kept at a constant temperature: 21°C, with a 16-hr:8-hr light:dark cycle) established at Cambridge University in 2005 and supplemented every summer thereafter with wild caught adults collected under licence from local field sites at Byron's Pool and Wicken Fen in Cambridgeshire.
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