To determine the osteoblast apoptosis, the culture medium was changed to a serum-free one with and without insulin or IGF-I, and total RNA or protein was collected after designated times for subsequent assays.
After designated time period, the culture density was measured by means of a haemocytometer (improved neubauer counting chamber) following the previously described method (Wu et al. 2005).
After designated time, the flasks contents were filtered, centrifuged (8000×g, 10 min) and residual dye concentration was monitored at respective wavelengths (HALO DB-20).The decolorization efficiency of MnP-I-PVAA and F-MnP for each dye was calculated using relation in Eq. (2).
The animals were sacrificed with CO2 asphyxiation followed by cervical dislocation at designated time points after tracer administration, and samples of tissues and body fluids were collected for radioactivity measurements and weighing.
At designated time points after inoculation, mice were euthanized by cervical dislocation under anesthesia.
Similarly, expression profiles of OE and Kan roots were compared at designated time points after inoculation.
At designated time points after injection of the radioconjugates (t=4 h, days 1, 2, 3, 5 and 7), groups of mice (n=3 5) were killed by Ethrane anaesthesia.
After cultivation without food for designated time (see below), animals were collected with NG buffer kept at 20 °C (except for Fig. S1 in Supporting Information) or at 23 °C (only for Fig. S1 in Supporting Information), and approximately 40 300 animals were placed at the center of the TTX plate.
At designated time intervals and after anesthesia, the animals (n = 4/time interval, n = 12 control, and n = 12 PNV-treated per age) were perfused transcardially with saline solution followed by 4% paraformaldehyde in 0.1 M PBS (phosphate buffered saline), pH 7.4.
Soon after the designated time, an election official began laboriously reading results, but only of six parliamentary races.
After a designated time, samples were collected and washed by distilled water, and then dried for analysis.
