2.4. Progranulin ELISA

Progranulin levels in dialysate and brain tissue were determined by ELISA (Adipogen # AG-45A-0019YEK-KI01) according to the manufacturer’s instructions. Microdialysis samples were diluted 1:1 with ELISA buffer prior to loading onto the ELISA plate. Brain tissue was prepared for ELISA by homogenizing in lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and centrifuging for 10 min at 5000 × g. Protein concentration of the supernatant was analyzed by BCA assay (ThermoFisher), and 40 μg of protein were loaded per well of the ELISA plate. ELISA signal was detected by reading absorbance using a Biotek Synergy LX plate reader. Concentrations of progranulin were determined based on a standard curve run on each plate.

To determine if the ELISA was capable of detecting granulins, 50 μg of recombinant mouse progranulin (Adipogen #AG-40A-0189Y) was cleaved overnight at 37 °C with 6 ng of recombinant human cathepsin L (R&D systems #952-CY-010) using a previously described protocol (Lee et al., 2017). For control samples, progranulin was incubated in reaction buffer without cathepsin L, or with cathepsin L and a protease inhibitor cocktail (Halt protease inhibitor cocktail, ThermoFisher #78429). After the reaction, complete cleavage was confirmed by SDS-PAGE on 10% polyacrylamide gels (Bio-Rad), followed by staining with Coomassie Fluor Orange (ThermoFisher). Samples were then diluted to 1 ng/mL progranulin in ELISA buffer and analyzed as described above.