F9 c 96 well plate fraction collector

Murine plasma was collected 45 min after a 1 mg/kg intravenous injection of Cy5-labeled siRNA conjugate into 6–8 week old, male CD-1 mice from Charles River (Wilmington, MA). Plasma was filtered (0.22 µm) and then injected into an AKTA Pure Chromatography System from Cytiva (Marlborough, MA) with three inline Superdex 200 Increase columns (10/300 GL). Fractionation was done at 0.3 ml/min using Tris running buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.2% NaN₃) into 1.5 ml fractions with a F9-C 96-well plate fraction collector (Cytiva). Cy5 fluorescence was measured in fractions (100 µl) in black, clear-bottom, 96-well plates from Greiner-Bio-One (Kremsmunster, Austria, REF 675096) on a SynergyMx from Biotek (Winooski, VT) at a gain of 120, excitation 642/9.0, emission 675/9.0. Fraction albumin-bound conjugate was determined by taking the sum of fluorescence intensity for fractions associated with albumin elution divided by the sum of fluorescence intensity for all fractions collected. Albumin-associated fractions were determined by running known protein standards through the SEC system and examining A280 of eluate from each of the fractions.