10.1371/journal.pone.0008058.g002 Figure 2  Formation of NB-like particles from protease-treated serum.
(A) NB-like particles cultured from protease-treated FBS. Stock solutions of 5% (w/v) trypsin or chymotrypsin were diluted into FBS at the final concentration of 0.5% (v/v), followed by incubation at 37°C for 2 hours. After incubation, the two protease-treated FBS solutions were inoculated into DMEM (columns labeled as “Trypsin” or “Chymotrypsin”) to the concentrations indicated on the top heading, starting from 0% (e.g. untreated, well 1) up to 10% (well 6). FBS was also treated with trypsin or chymotrypsin that had been inactivated by heat at 95°C for 1 hour (“Boiled Trypsin” or “Boiled Chymotrypsin”). For each treatment, A650 turbidity readings were performed within 1 hour following inoculation (“Day 1”) and after incubation in cell culture conditions for “1 Week” or “1 Month”, as indicated on the right. Untreated FBS (“None” column) or FBS that was treated with either boiled trypsin or boiled chymotrypsin produced a low bell-shaped curve of turbidity increase after 1 month of incubation. In contrast, inoculation of FBS that was treated with trypsin or chymotrypsin resulted in a more prominent straight dose-dependent turbidity increase after 1 month of incubation. (B) NB-like particles cultured from protease-treated HS were obtained as in (A). Notice the straight dose-dependent increase in turbidity, noticeable at “1 Month,” while both untreated HS and pre-boiled HS produced a low bell-shaped turbidity response. (C) Control experiment, performed with both untreated and boiled proteases inoculated directly into DMEM at the indicated % (w/v). No increase in turbidity was noticed for these control treatments following incubation.