Culture of NB-Like Particles from Protease-Treated Serum and Boiled Serum
Stock solutions of porcine pancreas trypsin (Sigma, St-Louis, MO, USA) or bovine pancreas chymotrypsin (Sigma) were prepared in water at a concentration of 5% (w/v). The protease solutions were sterilized by filtration through a 0.2-µm membrane. The NB-like particles depicted in Fig. 2 were prepared by adding trypsin or chymotrypsin into FBS or HS at a final concentration of 0.5% (v/v), followed by incubation at 37°C for 2 hours. A final volume of 1 ml of serum was used. Following incubation, the solution was diluted to final concentrations ranging from 0.1% to 10% (v/v) in DMEM and the mixture was incubated in cell culture conditions for several weeks. Alternatively, this experiment was repeated by treating FBS or HS with trypsin or chymotrypsin that had been boiled at 95°C for 1 hour. As a negative control, the stock solutions of trypsin or chymotrypsin were diluted into DMEM to final concentrations ranging from 0.01% to 3% (v/v) prior to incubation. The DMEM used for these experiments contained 0.02–0.2% sodium azide in order to prevent contamination.
To culture NB-like particles from boiled serum as shown in Fig. 3, HS was first diluted to 25% (v/v) using double-distilled water. FBS and 25% HS solutions were boiled at 95°C for 10, 30, or 120 min. The boiled sera were then diluted into DMEM to final concentrations ranging from 0.1% to 10% (v/v) and the solutions were incubated in cell culture conditions for several weeks. In some instances, stock solutions of 0.25 M CaCl2 and NaH2PO4 (both at pH 7.4) were successively added at a final concentration of 1 mM prior to incubation. The pH of the stock solution of 0.25 M CaCl2 was adjusted to 7.4 with 1 M HCl or 1 M NaOH whereas the pH of the NaH2PO4 solution was adjusted to 7.4 with 0.25 M Na2HPO4. These solutions were also sterilized by filtration through a 0.2-µm membrane prior to use. Parallel experiments were also conducted by adding aliquots from 0.25 M NaHCO3 pre-adjusted to pH 7.4, to the same final concentrations as those of CaCl2 and Na2HPO4. Results were virtually identical compared to experiments without carbonate. For brevity, only the data for the addition of calcium and phosphate are shown in the present study.