Generation of PINK1-deficient mice The PINK1 deficient mice were generated by Lexicon Genetics Inc. (The Woodlands, Texas, USA). See supplementary Methods S1 online. Cell culture Human NSCs (ReNcell VM NSCs) were provided by ReNeuron Ltd. Derivation of the line from human fetal ventral mesencephalon and maintenance has previously been described [39]. Briefly, NSCs were grown in laminin coated flasks/plates (Trevigen; 20 mg/ml in DMEM:F-12) in B27 medium (DMEM:F- 12 with 1X B27 supplement, 2mM Glutamine, 10 Units/ml heparin and 50 mg/ml gentamycin) containing growth factors human bFGF [10ng/ml, Peprotech] and human EGF [20ng/ml, Peprotech]. Media on NSCs was changed every 48 hours. Differentiation was carried out using either the stdD or preD method [39]. For StdD differentiation, NSCs were seeded at 30,000 cells/cm on laminin and expanded to 80% confluency in B27 medium containing growth factors (+GFs). Differentiation was initiated by changing to Differentiation medium: B27 medium without growth factors or antibiotics and supplemented with 1mM dibutyrl-cAMP (Calbiochem) and 2ng/ml GDNF (Peprotech). Media was not changed for the first 5 days, then after that every 3– 4 days. For the PreD method, NSCs were trypsinised and plated out at 30,000 cells/cm onto uncoated plates in B27 medium+GFs. NSCs were left to form aggregates for 7 days without a media change. Aggregates were then transferred to laminin-coated plates of equivalent surface area in B27 medium+GFs for 3–4 days to allow cells to disperse and reach 90% confluency. Differentiation was initiated and continued as for StdD protocol. ReNcell VM NSCs are referred to as human NSCs and ReNcell VM differentiated neurons are referred to as human midbrain neurons. SHSY5Y human neuroblastoma cells were maintained as described (Muqit et al 2006). For primary mouse cortical cultures, embryos were taken at gestational stage E16–E17. Animal husbandry and experimental procedures were performed in full compliance with the United Kingdom Animal (Scientific Procedures) Act of 1986. Cultures were set up as described in Supplementary Methods S1. Neurons were cultured in 96 well plates in maintenance medium [Neurobasal medium, 2% (v/v) B27 supplement, 2 mM gluta- mine, 100 I.U./ml penicillin and 100 I.U./ml streptomycin and 0.45% (v/v) D2(+) glucose]. Tissue harvested for biochemistry was snap frozen on dry ice and stored at 270uC. Embryos were obtained by crossing two heterozygote animals resulting in a mixture of F1 genotypes; genotyping is described in Supplemen- tary Methods S1. Apoptosis and cell death/viability Assays Annexin V PE binding. For analysis of apoptosis in NSCs and SHSY5Y cells, FACS was used to determine AnnexinV-FITC binding in conjunction with 7-Amino actiniomycin (7AAD) uptake according to manufacturer’s instructions (BD Pharmingen). Briefly SHSY5Y cells were seeded at 600,000 cells per well of a 6 well plate, in triplicate, and incubated overnight in SHSY5Y medium containing DMSO or 60 nM STS (Calbiochem). Human NSCs were seeded in triplicate in 6 well plates at 200,000 per well. The following day NSCs were incubated in media containing DMSO only (1:1000) or STS (50 nM) for 3 h at 37uC prior to processing. A total of 10,000 cells were analysed for cell death by FACS Calibur (Beckton Dickenson, CA) with the Cell Quest software. Multiparameter Cytoxicity Assay. We used the Multiparameter Cytotoxicity 1 HCS Reagent kit (Cellomics) for the assessment of overall culture viability using multiple cell PINK1 Deficiency parameters in differentiated human and mouse neurons at different time points. The assay was carried out according to the manufacturer’s instructions. Plates were analysed with an ArrayScan HCS reader and Cytotoxicity Indices (CIs) calculated using the Multiparameter Cytotoxicity 1 BioApplication Software (Cellomics). A full explanation of this algorithm is provided on the supplier’s website: http://www.cellomics.com/content/menu/ MP_Cytotoxicity/. Briefly, nuclear size, morphology and cell density measurements are made on nuclei automatically identified in fluorescence channel 1. The BioApplication then measures nuclear intensity in channel 2 and the intensity of lysosomal stain in the cytoplasmic region in channel 3. Upper and lower thresholds are automatically set for nuclear morphology/size, cell membrane permeability and lysosomal mass from user-configurable reference wells (control or wild type neurons). The percentage (indices) of cells in each well that are outside of these thresholds is ascertained. The Cytotoxicity Index is the maximal of nuclear morphology index, membrane permeability index, lysosomal mass index and cell density index, for each well. A mean CI is calculated based on three replicate wells per clone/genotype. Counting apoptotic nuclei. Human neurons were differentiated using the Pre-D protocol on poly-L-lysine glass coverslips (Biocoat) pre-coated in laminin. At 5 and 43 days differentiation (dd), cultures were incubated with either 1 mM STS or vehicle (DMSO) for 3 h before being fixed. Immunostaining was undertaken using dual labeling with anti-bIII tubulin, anti-TH and Hoechst 33342 as described [39]. Images were taken using an Axioplan Imaging microscope (Zeiss) using a 25X objective (see below). At least 50 adherent neurons per culture were examined and their nuclei scored as normal or pyknotic (fragmented/condensed) by a blinded observer. In addition, images from 5–10 non- overlapping fields of view were taken per culture using Axioplan software. Images were printed and the percentage of pyknotic versus normal neuronal nuclei were scored. Cytochrome C release. Release of cytochrome c from mitochondria in SHSY5Y cells was carried out using the Apoptosis Detection Kit (MitoSciences) according to manufacturer’s instructions. SDS-PAGE and immunoblot analysis For details see supplementary Methods S1 online. Cells were lysed by the direct addition of sample buffer and protein lysates separated on 10% or 16% Tris-Glycine mini gels by SDS- PAGE, before transfer onto nitrocellulose membranes. Mem- branes were incubated in non-fat milk in PBS with 0.1% Tween- 20 (PBST) then with primary antibodies (see Table S1). Membranes were washed then incubated with HRP-conjugated secondary antibodies (Dako). Membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and bands detected with x-ray films (BioMax). Equivalent protein loading was confirmed by stripping membranes and re-probing with anti-b-actin antibody. Band densitometry was performed using Quantity One 4.6.6 software (BioRad). RNA extraction, cDNA synthesis and RT-PCR Total RNA was isolated using TRIzol reagent and cDNA synthesised using random primers according to manufacturer’s instructions. cDNA was diluted 1:5 in water prior to PCR. All TaqMan Real Time PCR reactions were performed using using FAM-labelled PINK1 probe and VIC/TAMRA-dye labelled Endogenous control Human RPLPO or GAPDH (Applied Biosystems). PCR was performed using the ABI PRISM 7700 Sequence Detection System. Gene expression was calculated using the 2-ddCT method [40]. PLoS ONE | www.plosone.org 3 June 2008 | Volume 3 | Issue 6 | e2455