Pierce 660 nm protein assay with ionic detergent compatibility reagent

Brain tissues for immunoassays were resuspended in 200 µl of ice-cooled lysis buffer (50 mM Tris-buffer, 150 mM NaCl, 0.05% Tween-20, protease, and phosphatase inhibitor cocktail (1X)). Then, the proteins were extracted by sonication executing 40 cycles of 15 s on and 15 s off at 4 °C using a Bioruptor plus (model UCD-300, Diagenode). After lysis, samples were centrifuged at 4 °C, 20.000 g for 20 min, supernatants were collected and stored at −80 °C until further use. Total protein amount was determined using the Pierce™ BCA Protein Assay Kit according to the manufacturer instructions. Protein extraction of the hippocampal spheroids for proteomic analysis was performed as previously described (Pomeshchik Y et al., 2020). Proteins from the hippocampal graft and hippocampal post-mortem brain tissues were extracted using a lysis buffer of 25 mM DTT, 10 w/v% SDS in 100 mM Triethylammonium bicarbonate (TEAB). Samples were sonicated using 40 cycles of 15 s on/off at 4 °C in the Bioruptor plus (model UCD-300, Diagenode) after boiling at 99 °C for 5 min. Samples were centrifuged at 20,000 g for 15 min at 18 °C, and the supernatant was collected. Protein concentrations were measured using the Pierce™ 660 nm Protein Assay with ionic detergent compatibility reagent (Thermo) and preserved at −80 °C until further use.