Sh 9000

HeLa cells were transfected with 2 µg of plasmids and viable cells were assayed at 48 h after transfection by microplate reader SH-9000 (CORONA electric) using Cell Counting Kit-8 (cat. CK04; Dojindo Molecular Technologies, Kumamoto, Japan), a colorimetric assay, according to the manufacturer's instructions.

To address β2m amyloid fibril formation based on the supersaturation-limited mechanism, we measured the solubility of β2m monomer using an ultracentrifugation method combined with an ELISA assay^{37 (link),57 (link)}. First, β2m solution containing [β2m] = 1.0 mg/mL, [NaPi (pH 7.4)] = 20 mM, [NaCl] = 300 mM, [ThT] = 5 μM was prepared, and then amyloid fibril formation was induced by ultrasonication using the HANABI-2000 instrument. After a sufficient reaction time over 20 h, the solution reached an equilibrium state with amyloid fibrils, which was confirmed by the saturation of ThT fluorescence intensity. The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems). In the ELISA assay, optical density of the samples was recorded using a microplate reader (SH-9000, Corona Electric Co., ltd.) with SF6 software (Version 5.12.1, Corona Electric Co., ltd.).

Emission intensity of the purified proteins was measured using a micro-plate reader (SH-9000, Corona Electric). A final concentration of 5 μM coelenterazine-h was used as the luminescent substrate for these measurements. Experiments were performed at least in triplicate, and the averaged data were used for further analysis. Ca²⁺ titrations were performed by the reciprocal dilution of Ca²⁺-saturated and Ca²⁺-free buffers containing 10 mM MOPS, 100 mM KCl and 10 mM EGTA with or without 10 mM Ca²⁺ added as CaCO₃, at pH 7.2, 25 °C. The free Ca²⁺ concentrations were calculated using 0.15 μM for the apparent K_d value of EGTA for Ca²⁺. The Ca²⁺ titration curve was used to calculate the apparent K_d value by nonlinear regression analysis. The averaged data were fitted to a single Hill equation using Origin7 software (OriginLab).

Transporter activities were assayed according to previously described methods [23 (link)]. Briefly, CDFDA (10 μM), R123 (50 μM), and H33342 (10 μM), with or without their respective inhibitors (50 μM MK571, 50 μM verapamil, and 10 μM Ko143), were added 30 min before the HepG2 cells were treated with siRNA for 48 h. After a 1 h-incubation for CDFDA and H33342, or a 2 h-incubation for R123, the cells were washed twice with PBS. The cells’ fluorescence intensity was then measured by a fluorescence microplate reader (SH-9000, Corona Electric Co., Ibaraki, Japan) at Ex/Em wavelengths of 495/530 nm for CDF, 480/530 nm for R123, and 355/460 nm for H33342.

The thiocholine produced from the hydrolysis of acetylthiocholine by the endogenous AchE enzyme in each sample was quantified by a fluorescence colorimetric assay to examine the effect of ILA on AchE activity, which is a biochemical marker for neuronal differentiation in PC12 cells [28 (link)]. In brief, PC12 cells were grown and treated for five consecutive days, as mentioned. The cells were then lysed with ice-cold NP-40 cell lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA (pH 8.0) and 1% (v/v) NP-40. AchE activity of the cell lysates was determined by the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions. Assay signals were read with a fluorescence absorbance microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). at Ex/Em = 490/520 nm. The AchE activity was determined from the standard curve and normalized with the protein concentration in each sample. The protein concentration was determined by Pierce bicinchoninic acid (BCA) protein assay kit (Invitrogen, Paisley, UK) with bovine serum albumin as a standard.

The marine pennate diatom F. solaris JPCC DA0580 was isolated from the mouths of the Sumiyo and Yakugachi Rivers in Amami-Ohshima, Kagoshima, Japan [7 ,30 (link)]. The cells were cultured as previously reported [5 (link)]. Briefly, pre-cultivated cells containing approximately 40% lipid component were inoculated in half-strength Guillard’s ”f” medium (f/2) [31 (link)] at an initial density of 1 × 10⁵ cells/mL. The cells were grown at 25°C under continuous, cool-white fluorescent lights (140 μmol/m²/s). The cell density was estimated using a hemocytometer.
For the purpose of positive transformant selection, G418-resistant colonies were cultured with f/2 liquid medium containing 500 μg/mL G418 (Roche Applied Science, Indianapolis, IN), an aminoglycoside antibiotic, in a 96-well plate (Sumilon, Tokyo, Japan) at 25°C under constant illumination of 50 μmol/m²/s. After three weeks, 20 μL of the G418-resistant clone cultures were transferred to a fresh 96-well plate and incubated for another six days. In order to determine the cell density, the optical density (at 750 nm) of each well was measured in a microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). Furthermore, to evaluate the effect of glycerol on cell growth, wild-type and transformant strains were cultured in the presence of 10 mM to 500 mM glycerol.