1xb27 supplement

A thin film of liquid type I collagen (Advanced BioMatrix, San Diego, CA) was plated onto the bottom surface of 48-well Nucleon Delta-treated cell culture plate (Thermo Scientific, Waltham, MA) at a concentration of 100 ng/mL, incubated for 30 minutes, and then removed by aspiration. Human crypts were plated at a density of 500 crypts per well. These were grown as monolayers in Basic Medium with antibiotic-antimycotic (Invitrogen), 1 mM N-Acetylcysteine (Sigma), 100 ng/ml recombinant murine Noggin (PeproTech), 50 ng/ml recombinant murine EGF (PeproTech), 1xN2 supplement (Invitrogen), 1xB27 supplement (Invitrogen), 1 μg/ml recombinant human R-spondin 1 (R&D Systems, Minneapolis, MN), 10 μM Y-27632 inhibitor (Stemgent), 1 mM recombinant human Jagged-1 (R&D Systems), 5 μM CHIR99021 (GSK-3β inhibitor) (Stemolecule). Alternatively, crypts were suspended within Matrigel at a concentration of 100 crypts per 25 μl of Matrigel and plated in 3D on the 48-well Nucleon Delta-treated cell culture plate.

Mouse cortical primary neurons from P0 pups were cultured in Neurobasal medium supplemented with 1XB‐27 supplement and 1X L‐glutamine (Invitrogen, CA, USA) in a humidified atmosphere of 5% CO2 at 37°C. 293T cells, SH‐SY5Y cells, and doxycycline (DOXY)‐inducible mitochondrially targeting p53 (mito‐p53) cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum) and 1% penicillin/streptomycin (P/S) in a humidified atmosphere of 5% CO2 at 37°C.

Pups from heterozygous breeding were prepared at postnatal day 0 (P0). Brains were removed and transferred to a culture dish containing sterile HBSS (Gibco/Invitrogen). Brain hippocampi were dissected under a microscope and transferred to a 1.5 ml tube containing HBSS. For enzymatic digestion, the HBSS was replaced by 0.05% (w/v) Trypsin/EDTA (Gibco, 25300-054) and the tissue/Trypsin suspension was kept at 37 °C for 14 min (water bath). The tissue fragments were washed three times with 1 ml Neurobasal-A/Glutamax I media (Gibco/Invitrogen) and mechanically digested by trituration using a yellow pipet tip. The appropriate amount of the cell suspension was mixed with culture media (Neurobasal-A with Glutamax I (Gibco/Invitrogen), 1x B27 Supplement (Gibco/Invitrogen) and growth factors). The cells were seeded on poly-DL-ornithine hydrobromide (Sigma, P-8638) coated 96-well imaging plates (BD Falcon, 353219) or 6-well plates (Nunc). The media was replaced on the next day and then every two days. Cultures were maintained at 37 °C, 5% CO₂. The following growth factors were used for this study: rhFGF (5 ng/ml), rhBDNF (5 ng/ml), rmEpo (5 ng/ml) and rrCNTF (5 ng/ml), all from R&D Systems. To study physiological consequences of Lrrk2 deficiency in respect to insulin the culture media was supplemented with insulin-free B27 supplement (Gibco/Invitrigen).