PMC:2779105 / 182847-187338 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2779105","sourcedb":"PMC","sourceid":"2779105","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2779105","text":"Seeding of NB-Like Particles by Fetuin-A, Albumin, and Serum in Supersaturated Solutions\nStock protein solutions of BSF and HSA were prepared by dissolving the protein in HEPES buffer at a concentration of 10 mg/ml, followed by filtration through 0.2-µm filters. Several lots of proteins were used for the experiments described in this study. For Fig. 4, BSF and HSA were obtained from AppliChem (Boca Raton, FL, USA); for Fig. 5, BSF and HSA were from Sigma; and for Fig. 6, BSF was from Sigma while HSA consisted of a sterile solution used for intravenous injections (Plasbumin®-25; Talecris Biotherapeutics, Inc., Research Triangle Park, NC, USA). The stock solutions of proteins were then diluted into DMEM, individually or in combination, to concentrations varying between 0.7 µg/ml and 40 mg/ml. Other cell culture media obtained from Gibco were used in parallel, including Roswell Park Memorial Institute 1640 or RPMI-1640, F12 medium, medium 199, Glascow minimum essential medium, and Leibovitz L-15 medium. In some experiments, the precipitating reagents CaCl2 and NaH2PO4 were added to final concentrations varying between 0.1 mM to 1 mM. The final solution volume used was 1 ml. The solutions were then incubated in cell culture conditions for several weeks. The concomitant addition of carbonate, as outlined in the previous section, yielded similar results and data for such experimens are not shown here.\nTo culture NB-like particles from boiled protein solutions as shown in Fig. 7, solutions of BSF purified from FBS (Sigma) or BSA purified from FBS (Sigma) were prepared in HEPES buffer at a final concentration of 25 mg/ml. The protein solutions were filtrated through 0.2-µm membranes prior to use. These protein solutions were boiled at 95°C for 10, 30, or 120 min. The boiled protein solutions were diluted in DMEM at final concentrations ranging from 0.02 mg/ml to 2 mg/ml for boiled BSF and from 0.04 mg/ml to 4 mg/ml for boiled HSA. In some experiments, the precipitating reagents CaCl2 and NaH2PO4 were added successively each at a final concentration of 1 mM to the DMEM solutions containing proteins. The solutions were incubated in cell culture conditions for several weeks.\nIn order to evaluate the possibility that adsorbed proteins nucleate NB-like particles, BSF (Sigma), BSA (Sigma), or HSA (Talecris) were used in adsorption experiments. Adsorption of the proteins to polystyrene 24-well plates was performed by covering each well with 250 µl of protein solution at concentrations varying between 20 µg/ml to 10 mg/ml. Similarly, 250 µl of FBS and HS were also used at concentrations varying from 0.1% to 10%. The plate was incubated at 4°C overnight. Following incubation, the protein solution was removed and each well was washed twice with 250 µl of DMEM. We verified that the proteins used were adsorbed to the plate by staining the wells with 250 µl of Coomassie blue diluted 1∶5 in double-distilled water (Dye reagent concentrate; Bio-Rad Laboratories, Hercules, CA, USA). After 10 min of incubation, the shift to blue color was monitored directly by visualization of the plate and compared to controls without proteins which did not produce the blue color. Following adsorption of the proteins, 1 ml of DMEM was deposited in each well and the plate was incubated in cell culture conditions for several months. Alternatively, the protein solutions were deposited in each well and left to dry overnight under a laminar flow hood. Each well was washed twice with DMEM. 1 ml of DMEM was pipetted into each well and the plate was incubated in cell culture conditions. The coating agents poly-lysine (Sigma) and octadecyltrichlorosilane (OTS; Sigma) were also used to promote adherence of the proteins to the bottom of each well. For coating with poly-lysine, 250 µl of a 0.5% (w/v) solution of poly-lysine was incubated into each well. The plate was incubated for 5 min and the solution was removed. The plate was then incubated with the protein solution at 4°C overnight or left to dry overnight, followed by the same procedure described above. For coating with OTS, 250 µl of a solution of 0.5% (w/v) OTS was pipetted into each well and dried for 5 min. The protein solutions were then deposited into each well and the plate was processed as described above. Precipitation was monitored regularly by A650 turbidity readings, by visual inspection, and by using a cell culture inverted microscope (Diaphot; Nikon, Tokyo, Japan) at a magnification of 400X.","divisions":[{"label":"Title","span":{"begin":0,"end":88}}],"tracks":[]}