Neuronal loss and apoptosis measurements
Patient iPS-derived neurons exhibit HD-dependent phenotypes including
elevated caspase-3 signals and neuronal loss upon BDNF removal.
Fluorescent dye was used to measure the apoptosis phenotype, and
Immunostaining of Tuj1, a neuronal-specific-tubulin that capture
neuronal morphology, was used to measure neuronal shrinkage and loss
phenotype. The STHdh cells exhibit HD-dependent caspase 3 activity upon
stress, such as serum removal. For compound treatment in the cells, the
compounds were diluted in DMSO to 1X concentrations and added to the
cells at least one day after plating (for iPS-derived neurons, the
compounds were added one week after plating).The cells were stressed
(BDNF removal for iPS-derived neurons) 1 day after compound treatment,
and tested at the indicated time points. For caspase-3 activity
detection, NucView 488 caspase-3 dye (Biotium, #30029) was used for
staining as an indicator for apoptosis. For assaying the neuronal loss,
Tuj1, a neuronal specific tubulin was stained to show neuronal
morphology. Images were taken by Carl Zeiss microscopes and analyzed
blindly by ImageJ for caspase-3 and Tuj1 (Covance, #MMS-435P)
quantifications.
Western-blot analysis
Heala cells were plated at 75% confluency 24 wells plate. 16hr after
plating the cells, treatments were carried out as indicated
concentrations for 24hr. Then cells were lysed on ice in 6XSDS Sample
buffer (62.5 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 10% glycerol
and 0.01% bromophenol blue) for 30 min in presence of protease
inhibitors (Roche Diagnostics). Proteins were resolved by SDS-PAGE and
transferred onto a NC membrane (Millipore) by semi-dry Western- blot.
The membrane was blocked with 5% milk in PBST (1XPBS, 0.5% Tween20)
and incubated with the indicated primary antibody in 5% BSA in PBST
overnight at 4℃. The membrane was then washed three times using PBST and
incubated with the HRP-conjugated secondary antibody (Jackson
ImmunoResearch) diluted 1:10000 in 5% BSA with PBST for 1 h at RT,
washed again three times (5 min each) with PBST, and incubated with ECL
reagent mixture (Millipore). Chemiluminescence signals were detected
using the ChemiScope 3400 Mini (Qinxiang).