Terminal deoxynucleotidyl transferase dutp nick end labeling tunel

For the proliferation assay, passages 1–3 of primary neurospheres were dispersed to single cell suspension and were plated in Poly-D-Lysine/Laminin-coated dishes and left to stay for 24 h in complete medium. The day after cells were deprived of EGF/FGF for 3 h and then exposed for 24 h to 1 μM fingolimod-p in the presence or absence of 300 ng ml⁻¹ pertussis toxin (PTX) or 10 μM of ERK1/2 inhibitor U0126 that were added 30 min or 3 h before drug addition. At the end of the treatments, 10 mM BrdU (5-bromo-2′-deoxyuridine) was applied to the cultures for 16 h to label all actively proliferating cells before they were fixed and stained against BrdU. For the assessment of the effects of fingolimod on survival of NPCs after growth factors withdrawal, cells were plated as above and cultured for 72 h in EGF/FGF-free medium in the presence or absence of 1 μM fingolimod-p, with or without the addition of 10 μg ml⁻¹ anti-BDNF-neutralizing antibody. Finally, cells were fixed and stained with in situ cell death detection labeling kit (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL); Roche, Basel, Switzerland) according to the manufacturer's instructions.

Eukaryotic expression constructs for α-synuclein, 3flag-LRRK2 or eGFP-LRRK2 overexpression are described elsewhere [34 (link),60–63 (link)]. Untagged LRRK2 was generated by BamHI restriction-mediated excision of the 3flag-tag. LRRK2 knockdown constructs were cloned according to [64 (link)] and used with a blasticidin resistance marker (as in [34 (link)]). All transgenes were cloned in the pCHMWS backbone as described in [65 (link)]. A CMV promoter was used for all cell culture applications, while a CaMKII0.4 promoter was applied for in vivo overexpression purposes. All constructs were sequence confirmed. Lentiviral (LV) vectors were produced as described in Ibrahimi et al. [66 (link)]. Anti-α- or β-tubulin, vinculin and anti-FlagM2 antibodies are purchased from Sigma-Aldrich, anti-α-synuclein antibody from Enzo Life Sciences, LRRK2 P-S935 from Novus Biologicals and MJFF-2 from Abcam. Anti-eGFP antibody is generated in-house [67 (link)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and lactate dehydrogenase (LDH)-based viability kits are purchased from Roche. LRRK2 kinase inhibitor-1 (L2-IN1) [32 (link)] was purchased from Calbiochem and PF-06447475 [68 (link)] from Sigma-Aldrich.

Young (Y) and old hBM-MSCs (O) were cultured for 72 h under hypoxia conditions (0.1% O₂). Cell survival was determined using the Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. In brief, cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well. After incubation, 5 μl of CCK-8 reagent was added to each well. The absorbance was measured at 450 nm after 2 h of incubation at 37 °C.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; Roche, Laval, QC, Canada) was carried out according to the manufacturer’s instructions. The number of TUNEL⁺ cells in five randomly selected high-power fields per dish was determined and averaged with a Nikon Eclipase Ti fluorescent microscope. Cell apoptosis was expressed as the percentage of total cells (DAPI⁺).