Dulbecco s phosphate buffered saline dpbs buffer

Full-length, wildtype unmodified α-Syn was incubated at 37°C for one week under continuous shaking in an Eppendorf Thermomixer set at 600 r.p.m., to assemble into fibrillar form. 700 μM α-Syn was assembled in 50 mM Tris-HCl, pH 7.5, 150 mM KCl buffer (Table 1).
To prepare fibrils of full-length, phosphorylated, N-terminally acetylated, N-terminally acetylated and the E46K mutant α-Syn, recombinant protein (dialyzed and lyophilized) was diluted to 5 mg/mL in 200 µL of Dulbecco’s phosphate buffered saline (DPBS) buffer (Gibco; 2.66 mM KCL, 1.47 mM KH₂PO₄, 137.93 mM NaCl, 8.06 mM Na₂HPO₄-7H₂O pH 7.0–7.3). After 5 days of incubation at 37°C with constant agitation (1,000 rpm) in an orbital mixer (Eppendorf), reactions were sonicated for 5 min in a Branson 2510 water bath, aliquoted, and stored at −80°C. All fibrils were created in the presence of an air-water interface. The presence of amyloid fibrils was confirmed by thioflavin T fluorimetry and high molecular weight assemblies were visualized by gel electrophoresis.

IP1 production was detected by using the IP-One G_q HTRF kit (Cisbio). HEK293 cells were seeded into 24-well culture plates (Corning) at a density of 0.1 million per well and incubated overnight at 37 °C with 5% CO₂. Plasmids expressing wild-type H₁R or its mutants were transiently transfected using Lipofectamine™ 3000 regent (Invitrogen) when the cells reached 75% confluence. Twenty-four hours after transfection, the culture media was removed, and transfected cells were harvested and washed with Dulbecco’s phosphate buffered saline (DPBS) buffer (Gibco) twice and then resuspended in Hank’s balanced salt solution (HBSS) buffer (Beyotime) at a density of 1.0 × 10⁶ cells per milliliter. The 7 µL cell resuspension was seeded into a 384-well plate (Perkin Elmer) and incubated with 7 µL agonist or inverse agonist with various concentration gradients for 1 hour at 37 °C. Afterward, 3 µL IP1 d2 reagent and IP1 Tb cryptate antibody were added to the 384‐well plate and incubated for another 1 h at room temperature. Then, an EnVision multimode plate reader (Perkin Elmer) was employed to measure the HTRF ratio at 620/665 nm. The accumulation of IP1 was calculated according to a standard dose–response curve in GraphPad Prism 8 (GraphPad Software). Data are represented as the mean ± SEM, n = 3 independent samples.