*4.8. Human S100B ELISA Test*

The S100B protein concentration was determined in the incubation medium and in the cell lysates after 24 h of incubation with glucose or insulin and measured by adapting the enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer's protocols (human S100B ELISA Kit, Genorise, England). Briefly, the standards or samples (100 μL of lysates or supernatants) were added per well, and S100B was bound by the immobilized antibody during a 1 h incubation at room temperature. After triplicate washing with Assay Buffer (300 μL each) using an auto-washer (DiaWasher ELX50, Dialab GmbH, Austria), a detection antibody specific for human S100B (100 μL) was added to the wells and incubated for 1 h in RT. Following 3 washes with Assay Buffer (300 μL each), an HRP Conjugate (100 μL) was added for 1 h in RT. After a triplicate wash with Assay Buffer (300 μL each), a substrate solution (100 μL) was added to the wells, and color developed in proportion to the amount of S100B bounded in the initial step. The color development was stopped after 20 min by adding a stop solution. The intensity of the color was measured immediately using a microplate reader set to 450 nm with subtraction of readings at 540 nm or 570 nm to correct for optical imperfections in the plate. For each od ELISA experimental setting, the <sup>1</sup> × 104 cells were seeded in a 96-well plate. For intracellular concentration measurements, cells after incubation with glucose or insulin were centrifuged (3 min, 1200× *g*, RT), the supernatant was collected to Eppendorf vials. Cellular pellet was lysed according to freezethaw protocol and standardized for cellular protein concentration by BCA assay. Briefly, protein concentration was measured in cell lysates and adjusted to 50 μg protein per 100 μL. Such prepared samples were added per well and processed for ELISA assay.
