For the time course of MBG production, 6 ml of media conditioned by JEG-3 cells for 0 (baseline), 3 or 6 hours were extracted by 80% acetonitrile using Sep-Pak C-18 reverse-phase cartridges (Waters),9 (link) and the resultant extract was fractionated on an Agilent 1100 series HPLC system using Agilent Zorbax Eclipse XDB-C18 (Agilent Technologies, Palo Alto, CA), 4.6 × 150 mm, 5 µm particle size, 80 Å column, flow rate 1ml/min, in linear (10 – 85.5 %) gradient of acetonitrile against 0.1% trifluoroacetic acid (TFA) for 45 min.9 (link) Thirty 1.5-min fractions were collected and analyzed for MBG-immunoreactivity (MBG immunoassay, above).
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Time-course analysis of MBG production
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Other organizations : National Institute on Aging, National Institutes of Health
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Variable analysis
independent variables
- Time of media conditioning (0, 3, or 6 hours)
- MBG production
- Volume of media conditioned by JEG-3 cells (6 ml)
- Extraction method (80% acetonitrile using Sep-Pak C-18 reverse-phase cartridges)
- HPLC system (Agilent 1100 series)
- HPLC column (Agilent Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 5 µm particle size, 80 Å)
- HPLC flow rate (1 ml/min)
- HPLC gradient (linear 10 – 85.5% acetonitrile against 0.1% trifluoroacetic acid (TFA) for 45 min)
- Fraction collection (thirty 1.5-min fractions)
- Baseline (0 hours) as a control for time course of MBG production
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