Reagents, cell lines, and serum of STAM mice
Dulbecco’s modified Eagle’s medium (DMEM) and penicillin-streptomycin solution were obtained from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS) was purchased from Bovogen-Biologicals (VIC, Australia). Glyceraldehyde was purchased from Nacalai Tesque Inc. (Kyoto, Japan). The 5-[2,4,-Bis(sodioxysulfonyl)phenyl]-3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazole-3-ium (WST-8) assay kit and 3-[(3-cholamido-propyl)-dimethyl-ammonio]-1-propane sulfonate) (CHAPS) were obtained from Dojindo Laboratories (Kumamoto, Japan). The ethylene diamine-N,N,N’,N’-tetraacetic acid (EDTA)-free protease inhibitor cocktail was obtained from Roche Applied Science (Penzberg, Germany). C2C12 cells were obtained from KAC Co., Ltd. (Kyoto, Japan). The serum of STAM mice was purchased from SMC Laboratories, Inc. (Tokyo, Japan). The protein assay kit for the Bradford method was obtained from Takara Bio, Inc. (Otsu, Japan). A horseradish peroxidase (HRP)-linked molecular marker was obtained from Bionexus (CA, USA). A HRP-linked goat anti-rabbit IgG antibody was purchased from DAKO (Glostrup, Denmark). All other reagents and kits not indicated were purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). GA-derived AGE-bovine serum albumin (BSA) (TAGE-BSA), non-glycated BSA, and an anti-TAGE antibody were prepared as described previously [24].
Cell culture and cell seeds. C2C12 cells were incubated in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin under standard cell culture conditions (humidified atmosphere, 5% CO2, 37°C). Cells were seeded (1.9×104 cells/cm2) on 96-well microplates and culture dishes (Becton-Dickinson, NJ, USA).
Glyceraldehyde and aminoguanidine treatments of C2C12 cells. Glyceraldehyde was dissolved in phosphate-buffered saline (PBS) without Ca++ and Mg++ ((PBS)(-)), and then filtered before being added to C2C12 cells. The volume of PBS (-) (including glyceraldehyde) was 2.0 μL/100 μL of the total medium volume. All experiments were performed 24 h after treatments with 0, 0.5, 1, 1.5, and 2 mM glyceraldehyde. The cell culture method before the treatment with aminoguanidine (the volume of PBS (-) (including aminoguanidine) was 2.0 μL/100 μL of the total medium volume), an inhibitor of AGE production, was the same as that described above. Cells were pretreated with 0 or 8 mM aminoguanidine for 2 h followed by 0, 1.5, and 2 mM glyceraldehyde for 24 h (the volume of PBS(-) (including glyceraldehyde) was 2.0 μL/102 μL of the total medium volume).
Cell viability of C2C12 cells treated with glyceraldehyde and aminoguanidine. Cell viability was assessed using the WST-8 assay. Medium containing glyceraldehyde/aminoguanidine was removed and cells were washed with PBS (-). Ten microliters of WST-8 reagent was added to 96-well microplates in which C2C12 cells were cultured in medium (100 μL), and this was followed by an incubation at 37°C for 2 h in a CO2 incubator. Absorbance was measured at 450 and 655 nm using a microplate reader (Bio-Rad, CA, USA). Medium in the wells without cells was treated with glyceraldehyde/aminoguanidine, a medium change, and WST-8 reagent to measure background absorbance. Background absorbance was subtracted from experimental values.
Assessment of intracellular TAGE in C2C12 cells treated with glyceraldehyde and aminoguanidine using a slot blot analysis. This analysis was performed as described previously with some modifications [20-22]. Cells were washed with (PBS)(-) and then lysed in buffer [a solution of 2 M thiourea, 7 M urea, 4% CHAPS, and 30 mM Tris, and a solution of EDTA-free protease inhibitor cocktail (9:1)]. Cell extracts were then incubated on ice for 20 min, centrifuged at 10,000×g at 4°C for 15 min, and the supernatant was collected as the cell extract. Protein concentrations were measured using the protein assay kit for the Bradford method with BSA as a standard. Regarding the detection of TAGE, equal amounts of cell extracts, the HRP-linked molecular marker, and TAGE-BSA were loaded onto polyvinylidene difluoride (PVDF) membranes (0.45 μm; Millipore, MA, USA) fixed in the slot blot apparatus (Bio-Rad). PVDF membranes were cut to prepare two membranes and then blocked at room temperature (r.t.) for 1 h using 5% skimmed milk in PBS(-) containing 0.05% Tween 20 (skimmed milk-PBS-T). After this step, we used 0.5% of skimmed milk-PBS-T for washing or as the solvent of antibodies. After washing twice, membranes were incubated with (1) the anti-TAGE antibody (1:1,000) or (2) neutralized anti-TAGE antibody (a mixture of the anti-TAGE antibody (1:1,000) and 250 µg/mL of TAGE-BSA) at 4°C overnight. Membranes were then washed four times. Proteins on the membrane were incubated with the HRP-linked goat anti-rabbit IgG antibody (1:2,000) at r.t. for 1 h. After washing three times with PBS-T, membranes were moved into PBS(-). Immunoreactive proteins were detected with the ImmunoStar LD kit and band densities on the membranes were measured using the Fusion FX fluorescence imager (M&S Instruments Inc., Osaka, Japan). The densities of HRP-linked molecular marker bands were used to correct for differences in densities between membranes. The amount of TAGE in cell extracts was calculated based on a calibration curve for TAGE-BSA.
Analysis of serum TAGE levels in STAM mice. Briefly, each well of the 96-well microplate was coated with 1.0 μg/mL TAGE-BSA and incubated overnight in a cold room. Wells were washed three times with 0.3 mL of PBS containing 0.05% Tween 20 (PBS-T). Wells were then blocked by an incubation for 1 h with 0.2 mL of a solution of PBS containing 1% BSA. After washing with PBS-T, test samples (50 µL) were added to each well as a competitor for 50 µL of the anti-TAGE antibody (1:1,000), followed by an incubation at r.t. for 2 h with gentle shaking on a horizontal rotary shaker. Wells were then washed with PBS-T and developed with alkaline phosphatase-linked anti-rabbit IgG utilizing p-nitrophenyl phosphate as the colorimetric substrate. Results were expressed as TAGE units (U) per milliliter of serum, with 1 U corresponding to 1.0 μg of a TAGE-BSA standard as described previously [24]. Sensitivity and intra- and interassay coefficients of variation were 0.01 U/mL and 6.2 and 8.8%, respectively [25].
Non-glycated BSA and TAGE-BSA treatment of C2C12 cells and assessment of cell viability. C2C12 cells were treated with 0, 20 50, and 100 μg/mL of non-glycated BSA and TAGE-BSA, and then incubated for 24 h. Cell viability was measured using the WST-8 assay. The ratio of cell viability was calculated based on the viability of cells treated with TAGE-BSA versus those treated with non-glycated BSA.
Statistical analysis. Stat Flex (ver. 6) software (Artech Co., Ltd., Osaka, Japan) was used for statistical analyses. Data were expressed as means ± standard deviation (S.D.). When statistical analyses were performed on data, significant differences in the means of each group were assessed by a one-way analysis of variance (ANOVA). We then used the Bonferroni or Tukey’s test for an analysis of variance. P-values < 0.05 were considered to be significant.