Edta containing tubes

We collected 10-mL peripheral blood samples from all patients in the morning using EDTA-containing tubes (BD, USA), then mixed 7.5-mL blood samples with 6.5 mL of buffer and centrifuged the mixture at 8000 g for 10 min. The supernatant plasma was kept and mixed with buffer and immune-magnetic beads. After incubation at room temperature for 20 min, immune assay was performed. Excess reaction buffer and unresponsive magnetic beads were removed, and cells were washed from beads by elution buffer. Partial cell suspension liquids were mixed with APC-labelled CD45 antibody, PE-labelled CK8/18/19 antibody, and DAPI dye for staining. Excess dyes and antibody were removed. Stained cells were added into the MagNest sample chamber, which was incubated in the dark for 30 min. Within the MagNest sample chamber, cells dispersed into the analytical layer. CTCs were identified as CD45-CK+DAPI+. In morphology, CTCs showed clear cell contour, evenly distributed in plasma at more than 1 nucleus/cytoplasm ratio.

At baseline and 12 wk, a standard oral-glucose-tolerance test (OGTT) was administered following a 12-h overnight fast. Participants rested for ≥30 min prior to the insertion of a Teflon catheter into an antecubital vein for repeated blood sampling and remained patent by a 0.9% saline drip. After baseline blood collection, participants ingested 75 g glucose bolus (NOW foods) dissolved in 250 mL of water within 2 min (t = 0 min) (21 (link)). Venous blood was collected at the following time points: 0, 15, 30, 45, 60, 90, and 120 min after glucose ingestion into EDTA containing tubes (BD Biosciences). Blood glucose concentration was determined using a biochemical analyzer (YSI 2900 Life Sciences) in duplicate and subsequently centrifuged at 1000 × g for 10 min at 4°C. Aliquots of plasma were frozen and stored at –20°C until analyses. Plasma insulin concentrations were determined using a commercially available ELISA (ALPCO).
Plasma glucose and insulin concentrations were used to determine the Matsuda index and HOMA-IR according to established formulas (23 (link), 24 (link)). The Matsuda index was calculated by:

HOMA-IR was calculated by:

Further, the insulinogenic index (IGI) was utilized as a measure of β-cell function and calculated as ratio of insulin concentration at 30 min minus fasting insulin to the difference of glucose at the same time (25 (link)).

Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) containing tubes (BD, Franklin Lakes, NJ, USA). Plasma was isolated within 1 h using centrifugation at 1900 ×g for 10 min at 4°C to remove blood cells, and then at 16,000 ×g for 10 min at 4°C to remove additional cellular nucleic acid attached to cell debris. Plasma samples were transferred to RNase/DNase-free tubes and stored at −80°C.