Sf9 insect cells

Stable HEK293-mGlu₅ cell lines, generated as described previously,^{20 (link)} were maintained in complete DMEM supplemented with 5% FBS, 2 mM L-glutamine, 20 mM HEPES, 0.1 mM Non-Essential Amino Acids, 1 mM sodium pyruvate, and 500 μg/mL G418 at 37 °C in a humidified incubator containing 5% CO₂ and 95% O₂. Sf9 insect cells (Expression Systems) were maintained in a shaking incubator (120–140 rpm) at 27 °C in ESF921 growth medium (Expression Systems). Sf9 cells were infected with baculovirus to express full-length mGlu₅ and N-terminally truncated mGlu₅ constructs (see Supporting Information), to purify the mGlu₅ receptor with an N-terminal FLAG tag and C-terminal 8xHis tag (full details in the Supporting Information).

Sf9 insect cells (Expression Systems) were cultured at 27 °C in suspension in ESF921 media (Expression Systems). For baculovirus generation and passaging, this media was supplemented with 2% fetal bovine serum (FBS), while the media used to grow cells for protein production was not supplemented. HEK293T mammalian cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco), 100 units/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco). Tau seeding biosensor cell lines were generated by transducing with lentivirus generated from plasmid pMK1253 [48 (link)]. CHIP rescue in CRISPR KO lines was achieved by transducing with lentivirus expressing CHIP variants under control of the constitutive EF1a promoter and co-expression of nuclear-localized blue fluorescent protein (BFP).

A codon-optimized synthetic gene encoding residues 399-653 of the MLB1 capsid protein (NCBI YP_002290968.1) was cloned into the plasmid pBacPAK8 in frame with an N-terminal 10-histidine tag. A recombinant baculovirus stock was generated using the flashBAC system (Mirus bio). Sf9 insect cells (Expression Systems) in ESF-921 media were infected at a density of 2 million viable cells/mL with 0.025 mL of baculovirus stock/mL and cultured at 180 rpm, 27°C. Cells were harvested by centrifugation at 4 days post-infection, re-suspended in buffer A (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 2 mM MgCl₂, protease inhibitor cocktail (Millipore), and benzonase), and lysed via microfluidization. The lysate was clarified by centrifugation (40,000 x g), 0.22-um-filtered, and MLB1 spike was purified from the supernatant using TALON metal affinity chromatography. Fractions containing the MLB1 spike were dialyzed into 20 mM Tris-HCl pH 8.0, 20 mM NaCl and further purified using anion exchange chromatography. Fractions containing the MLB1 spike were dialyzed into 10 mM Tris-HCl pH 8.0, 150 mM NaCl, concentrated to ~ 5 mg/mL, flash frozen in liquid nitrogen, and stored indefinitely at −80°C.