A Novel Tauopathy 3D-Culture Model on a Biochip Figure 5. Scheme of tau mutation dependent cellular neurodegeneration mechanism based on observed relative impedance and cross section area alteration monitoring. Combination of time-dependent relative impedance (dark grey circles) and cross section area (light grey quad) alterations lead to a model of spheroid degradation that is in line with the obtained molecular and microscopic data. Comparing wildtype tau expressing spheroids incubated with 5 nM and 25 nM OA, hyperphosphorylation leads to an extended degeneration. More strikingly, comparison of wildtype tau (WT) and K280q tau (K280q) expressing spheroids reveals the tau-specific neurodegenerative processes. doi:10.1371/journal.pone.0049150.g005 Protein Chemical Analysis For protein extraction, spheroids were transferred in RIPA- buffer supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich) and sonificated (Hielscher GmbH). The protein concentration was determined using the Roti-Nanoquant Assay (Carl-Roth GmbH). 25 mg protein was treated with Laemmli sample buffer, separated on a 10% SDS polyacryl- amide gel and blotted on a PVDF membrane applying 2 mA/ cm2. Immunolabeling was realized using specific primary antibodies (p-tauT212, p-tauS422, p-tauS262, all three form Merck (1:1000); anti-neurofilament-L, SantaCruz, 1:1000; anti-b III- tubulin, SantaCruz, 1:1000; anti-synapsin I, Covance, 1:1000; anti-cleaved PARP [Asp214], BD Pharming, 1:1000; anti- caspase 3–cleaved tau [Asp421], SantaCruz, 1:1000) at 4uC overnight. Specific protein signals were detected applying a horseradish peroxidase-conjugated secondary antibodys (Dia- nova, 1:5000) via a chemiluminescence detection kit (MobiTec) and the ChemiDoc-XRS documentation system (BioRad). Membranes were stripped and reloaded with anti-tau human (Dako, 1:1000) and b-tubulin (Abcam, 1:1000), respectively to perform densitometric quantification using QuantityOne Soft- ware v4.6 (BioRad). Immunocytochemical Staining For cryodissection spheroids were fixed with 1 ml formaldehyde (4%) for one hour and transferred to 1 ml saccharose (25%) for several days. Spheroids were cryodissected (slices with a thickness of 20 mm) with a freeze microtome (Leica, CM3050S) and stored at 220uC. For immunocytochemistry, the slices were blocked with 3% BSA/0.1% Triton-X in PBS for 45 min and incubated with primary antibody (anti-neurofilament-200, SantaCruz, 1:500; p- tauS262, Merck, 1:500; anti-MC-1, kindly provided by Prof. Dr. Peter Davies from the Albert Einstein College of Medicine, 1:50) in blocking solution for two hours at room temperature. After three washing steps with PBS, the slices were treated with secondary antibody (Dylight-549, Dianova, 1:100) in blocking solution for one and a half hours at room temperature. Nuclei were stained with DAPI (Sigma, 1 mg/ml). Images were taken by laser scanning microscopy. Life Cell Imaging of SH-SY5Y 3D Cultures Expressing EGFP-fused Tau via Laser Scanning Confocal Microscopy Microscopic images of SH-SY5Y cells expressing EGFP-tau fusion protein were obtained by an inverse Nikon TE2000 microscope coupled with a Nikon digital eclipse C1plus confocal laser scanning module and the EZ-C1 3.1 Nikon spectral imaging software. EGFP was excited with the 488 nm Argon laser and detected with the 515615 nm band pass filter. All images were deconvoluted using 3D Huygens Deconvolution & Analysis Software v3.6 (SVI) and presented at maximum intensity projections. Determination of Spheroid Morphology and Cross Section Area Microscopic images of individual SH-SY5Y spheroids were taken every 24 hours after impedimetric measurement using a Nikon Eclipse TE2000U microscope. Spheroid cross section area was determined with the software ImageJ (National Institute of Health, USA) by manually marking spheroid contour. PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49150