Labassay glucose

Serum levels of biochemical parameters, glucose, cholesterol, triglyceride, and AST/ALT were measured using LabAssay^TM Glucose, LabAssay^TM Cholesterol, LabAssay^TM Triglyceride, and Transaminase CII-Test Wako. These kits were from FUJI Film Wako Shibayagi Cooperation (Shibukawa, Japan). Serum levels of HDL and LDL cholesterols were measured using HDL and LDL/VLDL Quantitation Kit (MAK045, Sigma-Aldrich, St. Louis, MO, USA).

Glucose and l-lactate concentrations in the medium were determined using LabAssay Glucose (Wako Chemicals GmbH) and l-Lactate Assay Kit (Colorimetric) (Abcam, Cambridge, UK), respectively.

Aged cells were first cultured for 5 days, and after harvest, cells were diluted 2-fold and cultured for another 9 days with 25 mL of 10% FBS-containing MEMα in 150 mm-diameter dishes. After passage, the medium was changed on culture day 4. The cells were harvested using trypsin-EDTA solution and stored with cryopreservation solution (STEM CELL BANKER, ZENOGEN PHARMA CO., LTD., Fukushima, Japan) in liquid nitrogen until use.
The CM was fractionated using the Amicon Ultra-2 mL (10 k-, 30 k-, 50 k-, and 100 k-device, Merck-Millipore, Burlington, MA, USA). Cell-proliferation-promoting activities were determined from three to eight independent plates.
Glucose concentration was determined colorimetrically with a clinical test kit (LabAssay™ Glucose, 638-50971, Fuji Film Wako, Tokyo, Japan).

Mouse blood samples were placed on ice for 1 h and then centrifuged at 3500 × g for 90 s. The blood serum was collected and total cholesterol (T-CHO), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), and triglyceride (TG) were measured using LabAssay™ Cholesterol (294-65801, Wako Pure Chemical Industries, Ltd, Japan), LabAssay™ Glucose (298-65701), LabAssay™ Triglyceride (290-63701), according to manufacturer’s instructions.

Streptozotocin (STZ) was used to prepare the diabetic mouse model. STZ is a nitrosourea analogue that causes insulin-dependent DM [8 (link)]. STZ (FUJIFILM Wako Pure Chemical Co., Ltd., Tokyo, Japan) was intraperitoneally (i.p) injected at 200 mg/kg of body weight dissolved in an ice-cold 0.1 M citrate buffer. Control mice received an equal volume of citrate buffer by i.p. injection. Blood glucose levels were measured with LabAssay^™ Glucose (FUJIFILM Wako Pure Chemical Co., Ltd.). One week after the STZ injection, DM was confirmed by the presence of hyperglycemia, and only mice with blood glucose levels >16.7 mmol/l were included in this study. Mice that did not become DM and mice weighing < 20 g at 10–12 weeks old were not used. We used insulin (Lantus insulin glargine, Sanofi-Aventis, Bridgewater, NJ) to temporarily decrease blood glucose levels and maintain glucose levels between 100 and 200 mg/dl. Insulin at a dose of 1.5 U/kg was subcutaneously injected 3 hours before measuring blood glucose levels. The dose of STZ and insulin was determined based on a previous report [9 (link),10 (link)].