Albumin and Hydroxyethyl Starch Purification

Albumin was purified from mouse serum (Kohsin Bio Ltd., Saitama, Japan) using the modified Cohn method [4 (link)] and then eluted from the column using 25 mM acetate buffer (pH 4.5) and neutralized to pH 7.2. Albumin was concentrated in centrifugal filter units with a nominal molecular weight limit of 50 kDa (Amicon Ultra-15 Centrifugal Filter Units; Merck, Darmstadt, Germany). Albumin purification was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined using the Bradford Ultra Total Protein Quantitation Kit (Abcam, Cambridge, UK) and adjusted to 5% (w/v) for administration. The albumin solution was stored at − 30 °C until further use.
HES powder (130/0.4/9; average molecular weight: 130 kDa; Fresenius Kabi, Bad Homburg, Germany) was used. HES labeled with FITC was prepared as previously described [4 (link)]. Low molecular weight (< 3 kDa) HES fractions were removed using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Units, Merck).