86 Chapter 4 distribution, using the Shapiro-Wilk normality test. Significant differences were detected using an unpaired t-test or one-way analysis of variance test. Dunnett’s post-hoc test was performed to analyze which groups differ significantly. Number of mice were calculated by power analysis and as a maximum 6-8 mice were used per group based on previous studies52. Data was considered significant when P < 0.05. BV2 and primary mouse macrophage culture All cells were kept at 37°C, 5% CO2 and 95% humidity. Mouse BV2 cells (a kind gift from Federico Roncaroli, Manchester) were cultured in RPMI-1640 containing 2mM GlutaMAX and 10% heat inactivated FBS (all Gibco). For experiments BV2 were seeded at 1x10^4 cells per well of a 96-well plate the day before treatment. Primary mouse bone marrow-derived macrophages (BMDMs) were obtained frombonemarrow of adult C57BL/6mice and cultured in DMEM containing 10% FBS, penicillin/streptomycin, and glutamine supplemented with M-CSF (10ng/mL; Peprotech) as previously described (Ying et al. 2013). All animal procedures were approved by the Memorial University Animal Care Committee in accordance with the guidelines set by the Canadian Council in Animal Care. Primary human macrophage culture All donors gave informed consent under a REC approved protocol (12/LO/0538). Human monocyte derived macrophages (MDMs) were obtained from fresh blood of male and female, healthy donors between 20 and 60 years after CD14-affinity purification. In brief, whole blood was diluted 1:1 with DPBS (Sigma), layered onto Ficoll (Sigma) and spun for 20 min at 800xg with minimal acceleration/deceleration. Peripheral mononuclear cells were collected, washed, and labelled with CD14-affinity beads (Miltenyi) according to the manufacturers protocol. CD14 monocytes were eluted and cultured at 5x10^5 cells/ml in RPMI-1640 containing 2mM GlutaMAX, 10% heat inactivated FBS, and 25ng/ml M-CSF (all Gibco) with medium change after 3 days. MDMs were used after 7 days in-vitro culture. For monocytes, M-CSF was omitted from the medium and cells were used immediately ex-vivo. Human TSPO genotyping Genotyping at rs6971 was performed by LGC. Where not specified, studies were performed with homozygous A carriers due to the high affinity for XBD-173 (high-affinity binders; HAB). Homozygous T carriers were grouped as low affinity binders (LAB). Heterozygous rs6971 carriers were omitted from this study. iPSC culture and microglia-like cell differentiation The human induced pluripotent stem cell (iPSC) line SFC841-03-01 (https://hpscreg.eu/cellline/STBCi044-A), previously derived from a healthy donor60, Oxford Parkinson’s Disease Centre/StemBANCC) was obtained under MTA from the James Martin Stem Cell Facility, University of Oxford and cultured in feeder-free, fully defined conditions. In brief, iPSCs were maintained in E8 medium on Geltrex (both Gibco) and fed every day until 80% confluent. For cell cluster propagation, iPSCs were lifted with 0.5 mM EDTA (Thermo) in DPBS and upon visible dissociation, EDTA was removed, and iPSC were diluted 4-6 times in E8 for culture maintenance. iPSCs were screened genotypically for chromosomal abnormalities using single nucleotide polymorphism analysis and phenotypically using Nanog (Cell Signalling) and Tra1-60 (BioLegend) immune positivity. Mycoplasma infection was excluded based on LookOut test (Sigma) according to manufacturer’s protocol. Microglia-like cells were differentiated
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