Analyses of purity and structural integrity of the AFP samples.

<p>A. Electrophoretogram of AFPI–III and AFGP samples provided by Nichirei Foods Inc. on 16.5% tricine-SDS-PAGE. The AFGP sample only contains molecules less than 10 kDa owing to a filtration step. B. The shape of an ice crystal and TH activity obtained for each AFP sample dissolved into the Euro-Collins solution. All protein concentrations were 10 mg/ml.</p

Time-dependent change in the survival rate (%) of rat insulinoma cell-line, RIN-5F, under hypothermic exposure (+4°C).

<p>A. The survival rate evaluated by counting the number of living cells (Fig. 1B). B. The survival rate evaluated by measuring the amount of insulin secreted from living cells (Fig. 1C). A freshly made Euro-Collins solution (EC) was used as a basic cell-preservation fluid to dissolve AFPI–III, trehalose, and BSA.</p

Flow-charts showing the preparation of RIN-5F cells and their survival rate evaluation.

<p>A. Preparation of RIN-5F cells before starting 1–5-day preservation experiments at +4°C. B. Procedures to estimate the number of living cells using the hemocytometer. C. Procedures to measure the amount of insulin secreted from the living cells. Medium-A consists of RPMI-1640 medium containing 10% of fetal bovine serum (FBS). Medium-B consists of the medium containing 1% of FBS. Trypsin-A consists of 0.25% bovine trypsin plus 1 mM of EDTA dissolved in phosphate buffered saline (PBS).</p

Confocal photomicroscope images of human hepatoma cells (HepG2) and rat insulinoma cells (RIN-5F) in Euro Collins-solutions containing 0.4 mg/ml of BSA (A), AFPIII (B and C), and AFPI (D) labeled with fluorescence.

<p>The images A and B show the slice data of 1 µm-width of HepG2, and A’ and B’ an intact cell images synthesized by stacking of the slices. These 4 images were reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073643#pone.0073643-Tsuda1" target="_blank">[22]</a> with permission. The images C and D are the slice data for RIN-5F cells. All the images were captured after 1 h-preservation at 37°C. Accumulation of AFPIII on the cell-surface is more evident compared with BSA.</p