Tween 20

The pGEX-2T/ERRα wt, Δ300aa, Δ398aa or pGEX-2T vector (GE Healthcare) was transformed into the BL21 strain (RBC Bioscience) and GST fusion protein expression was induced with 0.1 mM of IPTG (Nacalai Tesque). GST or GST-ERRα was purified using GSTrap™ FF (GE Healthcare). Whole cell lysates were incubated with GST-Accept (Nacalai Tesque) overnight at 4°C. Complexes were washed three times with washing buffer (50 mM Tris-HCl, pH 7.5) (Nacalai Tesque), 150 mM NaCl (Nacalai Tesque), 0.05% Tween20 (Wako)). Proteins interacting with ERRα were eluted with the sample buffer (2% SDS, 3% glycerol, 125 mM Tris-HCl, pH 6.5), 4% 2-mercaptoethanol, and 0.0125% BPB (all from Nacalai Tesque) and detected by western blot analysis.

PolyPs stored in, and released from, platelets were visualized using 4′,6-diamidino-2- phenylindole (DAPI; Dojindo, Kumamoto, Japan) [23 (link)]. In brief, platelets were immobilized on glass slides using a Cytospin 4 cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) and fixed with 10% neutral-buffered formalin for 1, 2, 4, or 18 h, or ThromboFix (Beckman-Coulter, Brea, CA, USA) overnight. After washing with PBS, the platelets were stained with six different DAPI-containing buffers (10 μg/mL) in the presence of Phalloidin-iFluor 555 (1:200 dilution) (Abcam, Cambridge, UK) for 30 min. PBS and Hanks balanced salt solution (HBSS) were mixed with 0.1% Tween-20 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 0.02% saponin, (Sigma-Aldrich, St. Louis, MO, USA), or 0.1% Triton X-100 (FUJIFILM Wako Pure Chemical) to create these six buffers. The specimens were then washed and mounted using an antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA) and subjected to microscopic examination using a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan) equipped with a BV-2A filter cube (excitation filter: 400–440 nm; dichroic mirror: 455 nm; barrier filter: 470 nm) to detect DAPI and phalloidin, respectively.

To test the dispenser, a 0.1% (w/v) safranine (196-00032, Wako Pure Chemical Industries, Ltd., Japan) aqueous solution was used as the working fluid. For the ELISA, Dulbecco's phosphate-buffered saline (DPBS) was prepared using a general method. Bovine serum albumin (BSA, A7030-50G, Sigma-Aldrich Japan Co., LLC, Japan) was dissolved in DPBS at a concentration of 1% (w/w) and used as a blocking buffer, sample/standard diluent, and conjugate diluent. Tween 20 (167-11515, Wako Pure Chemical Industries, Ltd., Japan) was added to DPBS at a concentration of 0.05% (v/v) and used as the washing solution. Phosphoric acid was diluted to 1 M with ultrapure water and used as a reaction stopper for a 3,3′,5,5-tetramethylbenzidine (TMB) peroxidase substrate (5120-0053, SeraCare Life Sciences, Inc.). In a demonstration of flow control of a multiplex ELISA device, 1% (w/v) safranine was dissolved in the sample/standard diluent, and 1 mM fluorescein (F6377-100G, Sigma-Aldrich Japan Co. LLC, Japan) was added to the wash solution for liquid visualization. The TMB was reacted with a small amount of horseradish peroxidase (HRP) and was colored.

Viability of the yeast cells on the electrode was examined by analyzing the membrane permeability changes with a live/dead backlight bacterial viability kit for microscopy and quantitative assays according to the manufacturer's recommendations (L7012, Molecular Probes, Eugene, OR, USA). To obtain dead yeast cells to act as a benchmark, we cultured yeast cells with 1% (v/v) Tween 20 (Wako, Osaka, Japan) containing PBS(−) for 60 min at 60ºC. After constant potential application, the yeast cells on the patterned working electrode were washed with 25 ml of PBS(−) and stained with the live/dead backlight bacterial viability kit for 20 min at RT (Fig. 1). After incubation for 20 min, the patterned working electrode was again washed with 25 ml of PBS(−) and observed using an epifluorescence microscope system (BX51, Olympus, Tokyo, Japan) connected to a digital camera (DP72, Olympus) and image analysis system software (DP2-BSW, Olympus).
For the measurement of yeast cell density on the patterned working electrode, the numbers of yeast cells on the electrodes were counted in random areas of 100 × 100 μm² after potential application. Measurement of yeast cell density was repeated eight times, and data were expressed as the mean of two independent experiments.

Protein samples were resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (GE Healthcare UK Ltd., Buckinghamshire, England) using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Membranes were stained with Ponceau S (Sigma–Aldrich Co. LLC., St. Louis, MO), and then blocked overnight with 5% skim milk (BD Biosciences, Franklin Lakes, NJ) in Tris-buffered saline with 0.1% Tween 20 (Wako) (TBST). Primary antibodies were diluted in 5% skim milk/TBST as following: anti-LPCAT3 (40 ng/ml), anti-FLAG M2 antibody (5 μg/ml, Sigma–Aldrich), anti-MTP antibody (50 ng/ml, BD), or anti-PDI antibody (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA). Horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) were used at a 1:2000 dilution in 5% skim milk/TBST. TBST was used for washing steps and changed at least three times between incubation steps. ECL select western blot detection system (GE Healthcare) was used for chemiluminescence, and detected using ImageQuant LAS500 (GE Healthcare).