Spectrophotometric microplate reader

Cells were plated at a density of 20,000 cells/well and urea levels were measured after 48 h of treatment. Urea assay was performed also with CHME-5 treated with TII for 72 h. In all these cases, urea levels were detected by the QuantiChrom Urea Assay kit (BioAssay System, Hayward, CA, USA) according to the manufacturer’s instructions. Two hundred μL Urea Reagent (BioAssay system, Hayward, CA, USA) were added to 50 μL culture medium, and the absorbance measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer Inc., MA, USA) after 50 min of incubation at room temperature under slow agitation conditions. A standard curve was generated in the range of concentrations 0–100 μg/mL using Urea as standard, and all the data were normalized proteins.

After 24 h from splitting, CHME-5 was plated at a density of 20,000 cells/well and treated for 72 h with TII, in medium containing 1%FBS, and different concentrations of Rapamycin (1–10 nM) or Sapanisertib (from 1 nM to 100 nM). After 72 h of incubation, medium was collected, and nitrite amount was measured in 80 μL of medium after adding 40 μL Griess Reagent (Sigma-Aldrich, St. Louis, MO, USA). The absorbance was measured at 550 nm in a spectrophotometric microplate reader (PerkinElmer Inc., Waltham, MA, USA). A calibration curve (0–100 μM) was generated using NaNO₂ (Sigma-Aldrich, St. Louis, MO, USA) as standard and all the data were normalized by protein quantitation (measured with Bradford’s method, using bovine serum albumin as standard).

Urea levels in IMhu cells were detected by the QuantiChrom Urea Assay kit (Cat. No.: DIUR-100—BioAssay System, Hayward, CA, USA), used according to the manufacturer’s instructions. Briefly after 24 h of incubation with the tested substances, an aliquot of cell culture media (50 µL) was mixed with 200 µL Urea Reagent (BioAssay system, Hayward, CA, USA) and the absorbance measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer Inc., Waltham, MA, USA). A standard curve was generated during each assay in the range of concentrations 0–100 µg/mL using urea as standard. In this range, standard detection resulted linear, and the minimum detectable concentration of urea was 3.12 µg/mL. The protein content in each sample was determined by Bradford’s method (Cat. No.: 5000006—Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA—Cat. No.: A2153—Sigma-Aldrich, St.Louis, MO, USA) as standard.

Urea levels in CHME5 and T98G cells were detected by the QuantiChrom Urea Assay kit (BIOassay System, Hayward, CA, USA), used according to the manufacturer’s instructions. Briefly, after 48 h of incubation with the B-CM and PS-CM, an aliquot of cell culture media (50 µL) was mixed with 200 µL Urea Reagent (Bioassay system, Hayward, CA, USA) and the absorbance measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer, Waltham, MA, USA). A standard curve was generated during each assay in the range of concentrations 0–100 µg/mL using urea as standard. In this range, standard detection was linear and the minimum detectable concentration of urea was 3.12 µg/mL. The protein content in each sample was determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using BSA as the standard.

Microglial viability was assessed by reduction of the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] contained in the CellTiter AQueous One Solution Reagent (Promega). For this assay, cells were seeded in 96-well plates. At the end of the experimental procedure, 20 μl of MTS reagent were added to the cells that were further incubated for 2 h. Living cells bio-reduces yellow MTS into a purple soluble formazan product with an absorbance peak at 492 nm, that was read in a spectrophotometric microplate reader (PerkinElmer Inc.)

Inducible nitric oxide synthase (iNOS) activity was assessed indirectly by measuring nitrite accumulation in the incubation media. Briefly, an aliquot of the cell culture media (80 μL) was mixed with 40 μL Griess Reagent (Merck, Darmstadt, Germany) and the absorbance was measured at 550 nm in a spectrophotometric microplate reader (PerkinElmer, Waltham, MA, USA). A standard curve was generated during each assay in the range of 0–100 μM concentrations using NaNO₂ (Merck, Darmstadt, Germany) as the standard. In this range, standard detection was linear and the minimum detectable concentration of NaNO₂ was 3.12 μM. In the absence of stimuli, basal levels of nitrites were below the detection limit of the assay at all the time points analyzed. The NO levels were normalized with the protein content determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (BSA) as the standard.