Protein quantification kit rapid

The purified rCpEF1α and rGST were concentrated by using Amicon Ultra filter units (50 K for rCpEF1α and 3 K for rGST) (Merck Millipore). The protein concentration was measured by using Protein Quantification Kit-Rapid (Dojindo laboratories, Kumamoto, Japan), and was diluted to 7.5 μM with elution buffer. Semi-confluent HCT-8 cells were washed twice, and treated with 500 μM EDTA in PBS for 5 min at 37 °C. Detached cells were washed with FACS buffer (PBS containing 2% fetal calf serum) by centrifugation at 1,500 rpm. Then 2 × 10⁶ washed HCT-8 cells were incubated with 100 μL of 7.5 μM rCpEF1α or rGST for 2 h at 4 °C, and then washed twice with FACS buffer. The cells were then incubated with 100 μL of rabbit α-GST antibody (Sigma-Aldrich) at 1:1000 dilution for 30 min at 4 °C, washed with FACS buffer containing 2% FCS twice, incubated with 100 μL of Alexa 488-conjugated α-rabbit IgG goat antibody at 1:1000 dilution for 30 min at 4 °C, and washed with FACS buffer again. As a negative control, detached HCT-8 cells which were incubated with neither proteins nor antibodies were also prepared. The sample was then analyzed on BD FACSVerse (BD Bioscience) using BD FACSuite software (BD Biosciences). HCT-8 cells were gated on forward/side-light scatter to distinguish them from debris. Cells (10,000 events) were analysed by Alexa 488 channels.

To determine levels of 8-OHdG, BDNF and IL-6 in the slice culture medium and the extracts of OBSCs, samples were analyzed using commercially available ELISA kits (New 8-OHdG Check ELISA kit, JaICA Co. Ltd. (Shizuoka, Japan), Human BDNF ELISA kit, RAB0026, Sigma-Aldrich, Mouse IL-6 ELISA Kit, RAB0308-1KT, Sigma-Aldrich, Tokyo, Japan). Culture medium was obtained during and after the cultivation of OBSCs. At the end of cultivation, OBSCs (2 slices) from a single culture membrane were carefully removed from the culture insert without containing matrigel. The OBSCs were then homogenized in 100 μL RIPA lysis buffer (WSE-7420 EzRIPA Lysis kit, ATTO Co., Tokyo, Japan) to obtain the extracts. Protein concentrations of the extracts were measured with a protein quantification kit (Protein Quantification Kit-Rapid, Dojindo Molecular Technologies, Inc., Kumamoto, Japan). For each ELISA, culture medium or OBSCs extracts (with same concentration of protein) were diluted to bring the expected concentration within the range of the standard curve and reacted with first and secondary antibodies, streptavidin-HRP and detection solution according to the manufacturer’s instruction. The reaction was stopped using the stop solution (from the ELISA kits) and absorbance read at 450 nm using a microplate reader (SH-9000Lab, Hitachi, Tokyo, Japan).

Tissue and HUVECs were homogenized in ice-cold Pierce RIPA Buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail cOmplete EDTA-free (Sigma-Aldrich), phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich), and sodium orthovanadate (Wako, Tokyo, Japan). Protein concentration was determined using a Protein Quantification Kit-Rapid (Dojindo Laboratories). Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63 (Abcam), CD9 (Abcam), CD81 (Cell Signaling Technology, Inc. Danvers, MA, USA), β-actin (Cell Signaling Technology), phospho-Akt (Ser473)(Cell Signaling Technology), Akt (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), and caspase-3 (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).

Frozen quadriceps muscles were powdered by mortar and pestle, lysed in ice-cold CelLytic M Tissue Lysis Reagent (C3228, Sigma-Aldrich) with a 1% protease inhibitor cocktail (25955-11, Nacalai Tesque, Kyoto, Japan) and 1% phosphatase inhibitor cocktail (07574-61, Nacalai Tesque), and centrifuged at 10,000g for 10 min at 4°C. C2C12 samples were homogenized in ice-cold CelLytic M Cell Lysis Reagent (C3228, Sigma-Aldrich) with the above-described protease inhibitor and phosphatase inhibitor cocktails. The protein concentration of the supernatant was measured using the Protein Quantification Kit-Rapid (PQ01, Dojindo, Kumamoto, Japan). Supernatant fractions of equal protein concentration were analyzed by Western blotting as described previously [6 (link)].