Lysis buffer

The NP tissues were cut into 1 mm³ pieces and extracted with lysis buffer (Cloud-Clone, China). After ultrasound treatment and centrifugation, the supernatant was obtained for the assay. The levels of cytokine LTα in human NP tissues and plasma samples were determined by a LTα-specific ELISA kit (Cloud-Clone). The detection range was from 15.6 pg/ml to1000 pg/ml, and the sensitivity limit was 8 pg/ml.

The collected blood was allowed to clot and then it was centrifuged at 1000× g for 10 min. Then, serum samples were collected into clean tubes and stored at −20 °C until used in ELISA assays. The isolated brain prefrontal cortexes were homogenized and lysed using a lysis buffer (Cloud-Clone Corp., Houston, TX, USA), following the manufacturer’s directions. Lysates were centrifuged at 10,000 rpm and 4 °C for 5 min and then they were stored at −80 °C until used. Protein content was determined by Bradford method [110 (link)]. The levels of IL-1β, IL-6, and TNFα, were quantified in a single serum or brain tissue sample using an appropriate for mice ELISA Kit (Cloud-Clone Corp., Houston, TX, USA), as previously described [59 (link)]. We performed the determinations in accordance with the manufacturer’s instruction. The optical density of individual wells was measured with a spectrophotometric microplate reader (BioTek, Elx808, Warsaw, Poland) at a wavelength of 450 nm. The concentration of cytokine in the samples was determined by comparing the optical density of the samples with the standard curve. Cytokine concentrations in the prefrontal cortex are expressed in picograms per ml/mg of protein, and picograms per ml of serum.

Protein extracts were obtained from samples by tissue homogenization in lysis buffer (Cloud-Clone Corp., USA) and centrifugation for 10 min at 2590×/g at 4 °C followed by 5 min at 5000×/g at 4 °C according to the manufacturer’s instructions. Protein concentration was determined by Bradford protein assay kit (Bio-Rad, Germany). Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the protein levels of GRN, NOTCH3, FN1, and PINK1. Standards, samples, reagents, and microplate preparations were performed as described by the manufacturer. Optical density of each well was measured using a microplate reader at a wavelength of 450 nm (Anthos Labtec Instruments, Austria). All conditions were measured in duplicates. Protein concentrations were calculated using the mean of the duplicates in relation to the total protein amount. The following kits were used: Progranulin ELISA Kit (No. E-EL-H1578, Elabscience, USA), Human Notch Homolog 3 ELISA Kit (No. SEL147Hu, Cloud-Clone Corp., USA), Quantikine ELISA Human Fibronectin (No. DFBN10, R&D Systems, USA), Human PINK1 ELISA Kit (No. MBS9327222, MyBioSource, USA).