NSC cell line generation
Multiple human induced pluripotent stem cell-derived neural stem cell lines (iPSC-NSCs) were obtained from multiple vendors and tested for basal NSC maintenance and intrinsic neuronal differentiation quality in a small scale (Axol, Millipore, ThermoFisher, MTI global, Tempo Bioscience, Roche). iPSC NSC from MTI Global Stem (HIPTM Neural Stem Cells, BC1 line) and NSC-line2 (gift from Christoph Patsch, Roche (Basel, Switzerland) were chosen based on the following criteria: (1) able to be maintained a homogenous NSC morphology beyond 40 passages; (2) >80% neuronal differentiation efficiency; and (3) fast growth rate at least 1:3 expansion/split ratio. (4) No remaining progenitor cells after differentiation. NSCs were transfected to stably and express inducible ASCL1 and NGN2, transcription factors whose expression has been shown to increase differentiation efficiency in combination with differentiation media (Bardy et al.28; Ladewig et al.30; Zhang et al.31). Transcription factors ASCL1, NGN2, and EGFP were cloned into a vector containing a cumate-inducible promoter (Systembio), then stably transfected using Neon electroporation. Both cell lines were cultured according to the manufacturer’s instructions. Briefly, cells were cultured on flasks coated with a 1:100 Geltrex (ThermoFisher) solution for at least 1 h in a 37 °C cell incubator. Cells were grown in Neural Stem Cell Growth Media and 0.75 μg/ml Puromycin selection marker 37 °C 5% CO2 cell culture incubator. Cells were passaged every 3–4 days when confluent using TrypLE Express Enzyme (Gibco) and split at no more than a 1:3 ratio depending on cell density.
Astrocyte culture
Primary human astrocytes (ThermoFisher) were cultured and passaged according to manufacturer’s instructions in Primary Human Astrocyte Medium on T-650 flasks coated with 1:100 Geltrex (ThermoFisher) solution for at least 1 h in a 37 °C cell incubator. Full volume of media was changed every 3–4 days until cells were confluent. Cells were passaged when confluent using TrypLE Express Enzyme (ThermoFisher) and split at a ratio of up to 1:6.
For astrocyte validation experiments, primary human astrocytes were detached using Accumax (Innovative Cell Technologies) and seeded onto 384-well CellCarrier Ultra imaging plates (PerkinElmer) coated with 1:100 Geltrex (ThermoFisher) solution for at least 1 h in a 37 °C cell incubator. Cells were seeded at a density of 2000 cells/well in Neuron Maintenance Medium. Cells were placed in a 37 °C cell incubator for 24 h to allow for attachment. Aβ42 and antibody treatments were added according to “Aβ42 Oligomer Experiments”. Astrogliosis was validated by immunostaining of the following markers: Guinea Pig anti-GFAP (Synaptic Systems 173004 1:500), Rabbit anti-EAAT1 (Boster PA2185, 1:500), Rabbit anti-vimentin (Cell Signaling 3932S 1:500), Rabbit ALDH1L1 (Abcam ab19029, 1:500).
NSC differentiation
NAG-NSCs were grown until confluent and then detached using TrypLE Express Enzyme (Gibco) and plated onto a T-650 flask coated with 50 μg/ml poly-D-lysine (Sigma-Aldrich) and 10 μg/ml mouse laminin (ThermoFisher). Cells were plated at a density of 0.7 × 108–1.0 × 108 cells/flasks in Neuron Differentiation Media supplemented 100 μg/ml Cumate (System Biosciences), 1 μM PD0332991 cell cycle inhibitor (Tocris), and 10 μM Y27632 Rock inhibitor (Tocris). Cells were differentiated for 5–7 days with one half volume differentiation media changed every 3 days.
Neuron replating
Once differentiated, cells were detached using Accumax (Innovative Cell Technologies) supplemented with 5% trehalose dihydrate (Sigma-Aldrich), 1U Papain (Worthington), 10 μM Y27632 (Tocris), 8 mM kynurenic acid (Sigma-Aldrich). Cells were plated using Tecan Fluent automation workstation into 384- or 96-well CellCarrier Ultra imaging plates (PerkinElmer) coated with 50 μg/ml poly-D-lysine (Sigma-Aldrich) and 20 μg/ml recombinant human laminin (Sigma-Aldrich) in Neuron Differentiation Media supplemented with 10 μM Y276342 Rock Inhibitor (Tocris), and 1X RevitaCell (Gibco).
Astrocyte coculture
Astrocytes were added to neuron culture (5–10 days after neuron replating). Astrocytes were detached using Accumax (Innovative Cell Technologies) and plated using Tecan Fluent automation workstation into 384- or 96-well plates containing differentiated and replated neurons at a density of 4000 or 20,000 cells/well, respectively.
Neuron–astrocyte coculture automated maintenance
Neurons and astrocytes were cultured in 384- or 96-well Cell Carrier Ultra plates (PerkinElmer) in Neuron Maintenance Medium and half of the volume of culture media was changed using Tecan Fluent automation liquid-handling workstation every 3–4 days for at least 8 weeks and up to 6 months before subsequent experimentation. Tecan Fluent was programmed to utilize its features of automated tip loading, lid removal, and to aspirate half the volume of culture media and add new culture media for up to 30 plates at a time. Barcode-operated plate storage incubator technology was integrated into the system for plate organization and retrieval.
iPSC microglia culture
iPSC-derived iCell microglia were obtained from multiple vendors and screened for microglia marker expression. Cells from two vendors were used in this publication. iPSC microglia from Cellular Dynamics international were differentiated based on published protocols65. BrainXell Inc microglia differentiation was based on published protocol. Briefly, iPSCs were treated with BMP, FGF, and Activin for 2–4 days to induce mesoderm fate, then treated with VEGF and supportive hematopoietic cytokines for 6–10 days to generate hematopoietic progenitors. HPCs were seeded onto Matrigel-coated flasks, treated with IL-34, IDE1 (TGF β1 agonist), and M-CSF for 3–4 weeks to differentiate into microglia. Human iPSC microglia were validated by immunostaining of the following markers: Goat anti-TREM2 (1:500, R&D Systems, AF1828), Mouse anti-MERTK (1:500, Biolegend, 367602), Rabbit anti-IBA1(1:1000, Wako Chem, 019-19741), Rabbit anti-TMEM119 (1:500, Abcam, ab209064), CD33 (1:500, Biolegend, 303302), CX3CR1 (1:500, BioRad, AHP1589), CD64 (1:500, Biolegend, 305012), P2RY12 (1:500, Sigma, HPA014518), CD32 (1:500, Biolegend, 303212), PU.1 (1:500, Cell Signaling, 266S).
Frozen Cells were thawed and immediately seeded at a density of 8000 cells/well of a 384-well plate onto an 8-week-old neuron–astrocyte coculture in Microglia media. BrainPhys neuronal media (Stem Cell Technologies) supplemented with 1X B27 with vitamin A (ThermoFisher), 1X N2 Plus media supplement (R&D Systems), 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 mM creatine (Sigma-Aldrich), 200 nM L-ascorbic acid (Sigma-Aldrich), 1 μg/ml mouse laminin (ThermoFisher), 0.5 mM glutamax (ThermoFisher), 0.5X penicillin-streptomycin (ThermoFisher), 1X Normocin (Invivogen), 5 ng/ml TGF-b (Peprotech), 100 ng/ml human IL-34 (Peprotech), 1.5 μg/ml cholesterol (Sigma-Aldrich), 1 ng/ml gondoic acid (Cayman Chemicals), 100 ng/ml oleic acid (Cayman Chemicals), 460 μM Thioglycerol (Sigma-Aldrich), 1X Insulin-Transferrin-Selenium (ThermoFisher), 25 ng/ml rhM-CSF (Peprotech), 5.4 μg/ml Human Insulin Solution (Sigma).
Monocyte-derived macrophage culture
Monocyte-derived human macrophages were obtained from PromoCell. Macrophages were differentiated into M1 or M2 polarized macrophages by treatment with GM-CSF and M1 Macrophage Generation Medium or M-CSF and M2 Macrophage Generation Medium, respectively (PromoCell). Cells were differentiated for 9 days, then frozen into aliquots. Frozen Cells were thawed and immediately seeded at a density of 8000 cells/well of a 384-well plate onto 8-week-old neuron–astrocyte coculture in Microglia media (above) supplemented with GM-CSF or M-CSF before subsequent experiments.
Soluble Aβ species generation
AggreSure™ β-amyloid (1–42), Human monomers were purchased from Anaspec and resuspended in DMSO followed by PBS to form a 100 μM solution. Aβ oligomerization was made following a previously published protocol (Stine et al., 2011). Briefly, Aβ42 monomers were subsequently incubated at 4 °C for 24 h, then frozen at −80 °C to stop the oligomerization process. We found that neuronal toxicity is variable between lots, but is consistent within one lot. Five to six lots of Aβ42 monomers were screened at a time and assessed for neurotoxic and the degree of toxicity. The working lot was purchased in bulk (50 mg). During this study in the span of 4 years, 3 rounds of screening were conducted after the bulk working lot of abeta was used up. Total of three lots were purchased in bulk for this study. We noted the variability is improved with GMP versions of Aβ monomer. For fluorescent Aβ42 oligomer experiments, β-amyloid (1–42), HiLyte™ Fluor 555-labeled, Human (Anaspec) was used. For pHrodo experiments, β-amyloid (1–42), Human was labeled with pHrodo™ Green AM Intracellular pH Indicator (Invitrogen) according to manufacturer’s protocol.
Soluble Aβ species experiments
Prior to experimentation, media volume in wells containing neurons was equalized with liquid-handling automation (Bravo) in order to ensure precise control of concentrations. All Aβ42 oligomers, anti-Aβ, small molecules, inflammatory cytokines were prepared at 10× concentration then added to neuronal culture media at an appropriate volume to ensure the final concentration listed. For the repeated dosing experiments, the media were refreshed 50% at each dosing first, before adding Aβ42 oligomers, and/or anti-Aβ antibody at the specified final concentration.
Aβ42 oligomer conformation ELISA
To determine the oligomeric species’ of Aβ42 present in sAβ42s, we developed oligomer selective and fibril selective ELISAs. To detect the presence of oligomeric Aβ42, we utilized a 6E10-6E10 assay utilizing the same anti-Aβ42 (6E10) as both capture and detection to selectively bind to oligomeric species containing more than one exposed 6E10 binding site. To further test for oligomeric species, we developed a GT622-6E10 assay using Aβ-oligomer specific antibody (GT622) as capture, and pan-Aβ antibody (6E10) as detection. Finally, we tested for the presence of fibril species using Aβ fibril selective antibody clone (OC) as capture and pan-Aβ antibody (6E10) as detection. sAβ42s to be tested were prepared according to “Aβ42 Oligomer Generation” protocol. Clear, flat bottom immuno nonsterile maxisorp 384-well (Nunc) were coated with 100 ng/ml of different anti-Aβ42 antibody (6E10, Biolegend, 803003; GTX622, Genetex, GTX635160; OC, Millipore, AB2286) in 0.05 M sodium carbonate buffer, pH 9.6 overnight. Plates were washed 3× with 0.05% Tween-20 in 1X PBS, then blocked in 0.5% BSA + 15PPM Proclin in 1X PBS, pH 7.4 for 1 h. All samples were quantified against Aβ42 monomer diluted to 1 μg/ml in 0.5% BSA + 0.05% Tween-20 + 0.35 M NaCl + 0.25% CHAPS + 5 mM EDTA in 1X PBS, pH 7.4 (Assay Buffer), then diluted twofold to 15.625 ng/ml. Sample Aβ42 oligomers were diluted to 1 μg/ml in Assay Buffer, then diluted threefold to 37 nM. Blocked plate was then washed 3× in 0.05% Tween-20 in 1X PBS, then samples, standards, and controls were added and incubated at 4 °C overnight. After sample incubation, plate was washed 6× with 0.05% Tween-20 in 1X PBS, then 100 ng/ml conjugate antibody in Assay Buffer (6E10, Biologend, 803003) was added and incubated for 1 h at RT. After incubation, plate was washed 6× with 0.05% Tween-20 in 1X PBS, then Strep-Avidin Poly 80 HRP detection antibody was added at a dilution of 1:10,000 in Assay Buffer and incubated for 45 min at RT. After incubation, plate was washed 6× with 0.05% Tween-20 in 1X PBS, and TMB substrate was added to each well, then incubated for 10–15 min. After appropriate color was developed, 1 M H3PO4 was added to quench the reaction. Finally, plate O.D. was read at 450–630 nm.
APOE genotyping
Neuronal cells to be genotyped were harvested and lysed according to “Mammalian Cells Lysate” instructions included in PureLink™ Genomic DNA Mini Kit (Invitrogen). Genomic DNA was purified according to manufacturer instructions included in PureLink™ Genomic DNA Mini Kit (Invitrogen). DNA was quantified using Nanodrop spectrophotometer, then diluted to 15 ng/µL.
APOE genotyping PCR was performed using EzWay Direct ApoE Genotyping Kit (Koma Biotech), which includes a proprietary ApoE primer mixture for E2 (Cys112/Cys158), E3 (Cys112/Arg158), and E4 (Arg112/Arg158). Fifteen nanograms of purified DNA for each sample was added to a 25 µL sample reaction. Primers PCR conditions and cycling was performed as follows:
Initial denaturation at 95 °C for 15 min.
Denaturation at 95 °C for 30 s (35 cycles).
Annealing at 65 °C for 30 s (35 cycles).
Extension at 72 °C for 1 min (35 cycles).
Final extension at 72 °C for 10 min.
Amplified DNA was run on a 1% agarose gel at 100 V for 1 h, then imaged.
The genotype of iPSC-NSC neurons from MTI Global Stem is ApoE4/4, while the NSC-line2 is ApoE3/3. We observed similar phenotypes in both of these cell lines, and thus ApoE genotype status does not appear to affect the phenotypes reported throughout this study. Human primary astrocytes (ThermoFisher) and iPSC microglia (CDI and BrainXell) are ApoE 3/3 (Table 1).
Immunofluorescence staining
Cells were fixed with 4% PFA, 4% sucrose at room temperature for 20 min using Bravo automation. Fixed cells were then washed two times with PBS using Biotek 406 microplate washer (Beckman Coulter), followed by permeabilization and blocking by incubation with a solution containing 1X PBS, 0.1% Triton X-100, 2% donkey serum, and 1% BSA at room temperature for 30 min. Blocking solution was removed and cells were incubated with primary antibodies in blocking solution overnight at 4 °C. After washing six times with PBS on Biotek 406 cell plate washer (Beckman Coulter), cells were then incubated with fluorophore-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc.) for 1 h at room temperature in the dark to avoid photobleaching. Cells were then washed six more times with PBS before imaging. Fluorescent images were captured using an InCell6000 confocal microscope (GE Healthcare Life Sciences). The image analysis was performed with InCell 6000 analysis software.
Small-molecule focused screen and IC50 curve follow-up
A focused library of 70 small molecules and natural products (Genentech, Selleckchem, Tocris Biosciences) implicated with neuroprotective properties were tested using a cell rescue assay in a 384-well microplate format (Cell Carrier Ultra). For double culture screen, mature human neuronal cultures (12 weeks+) were equalized in volume immediately before experiments using Bravo Automated liquid-handling platform (Agilent) to account for evaporative edge effects. For the triple culture screen, mature human neuron cultures (12 weeks+) were equalized in volume using Bravo Automated liquid-handling platform (Agilent), then microglia were plated according to the “iPSC Microglia Culture” section. After 3 days and immediately before experiments, triple culture volumes were equalized using Bravo Automated liquid-handling platform (Agilent) to account for evaporative edge effects. For both double and triple culture screens, pharmacological treatments were prepared from 10 mM DMSO stocks. 10 mM drug stocks were first diluted into Neuron Maintenance media to create intermediate stock solutions ranging from 1 to 1000 μM. Double screen drug concentrations used: 50, 25, 12.5, 6.25 μM. Triple screen concentrations used: 50, 12.5, 3.125, and 0.78 μM. Triple screen concentration was reduced to lower drug toxicity and increase hit rate. Intermediate stocks were then diluted to create 10X concentrations of drugs, which were serially diluted to generate a range of concentrations necessary to generate a dose–response profile. Final DMSO in treatments was kept at below 0.1%. Abeta oligomers were added to wells ~1 h after drug treatment to a final concentration of 5 μM Abeta oligomer per well. Cells were incubated with compounds and Abeta for 3–7 days (depending on Abeta lot strength and pharmacological profile of drugs tested) at 37 °C and 5% CO2. Cells were fixed with 4% Paraformaldehyde solution (Electron Microscopy Sciences), 4% sucrose solution. Fixed cells were stained for various neuronal markers for dendrites (Map2), synapse (Synapsin1/2), nucleus (CUX2), and axons (Tuj1). Plates were imaged using the InCell6000 confocal microscope (GE) and then analyzed with the InCell 6000 image analysis software.
Immunostaining antibodies
The following primary antibodies were used for immunostaining experiments: Chicken anti-MAP2 (1:2000, GeneTex, GTX85455), Guinea pig anti-Synapsin 1/2 (1:750, Synaptic Systems, #106 004), Mouse anti-Tau HT7 (1:500, Invitrogen, MN1000), Mouse anti-Aβ 6E10 (1:500, Biolegend, #803003), Rabbit anti-CUX2 (1:500, Abcam, ab140329), Rat anti-CTIP2 (1:500, Abcam, ab19465), Guinea pig anti-vGlut2 (1:1000, Synaptic systems, #135304), Mouse anti-Shank (1:200, Millipore N23B/49), Mouse anti-PSD95 (1:200, Millipore, K28/43), Mouse anti-GluR1 (1:200, Millipore), Mouse anti-PanShank (1:200, Millipore, N23B/49), Mouse anti-GluR2 (1:200, Millipore), Mouse anti-PanSAPAP (1:200, Millipore), Mouse anti-NR1 (1:200, Millipore), Rabbit anti-phospho-Tau S396-404 (1:3000, Genentech), Rabbit anti-phospho-Tau S235 (1:1000, ThermoFisher), Mouse anti-β-Tubulin Tuj1 (1:500, Biolegend, #801202), Chick anti-Neurofilament-Heavy Chain (1:3000, Abcam), Rabbit anti-Iba1 (1:1000, Wako Chem, 019-19741), Rabbit anti-TMEM119 (1:500, Abcam, ab209064). Rabbit anti-ApoE (1:500, ThermoFisher 16H22L18), Rabbit anti-APP (1:500, Abcam ab32136), Guinea pig anti-GFAP (1:500 Synaptic System 173004), Rabbti anti-ALDH1L1 (1:500, Abcam ab190298), Rabbit anti-Vimentin (1:500, Cell Signaling 3932S), Rabbit anti-EAAT1 (1:500, Boster PA2185).
Secondary antibodies were obtained from Jackson ImmunoResearch. Whole antibodies made in a donkey host with IgG (H + L) specificity and minimal cross-reactivity were used. For immunostaining experiments, secondary antibodies against their appropriate primary antibody species and conjugated to either DyLight405, AlexaFluor488, Cyanine Cy3, or AlexaFluor647. All 405- and 488-conjugated antibodies were used at 1:200, all cy3 conjugated antibodies were used at 1:600, and all 647-conjugated antibodies were used at 1:800.
Image analysis
Image analysis was performed using supplied InCell6000 analysis software, IN Cell Software was utilized to segment cellular regions of interest such as dendrites, cell bodies, and axons. During image acquisition, 9 images are collected per well, which are then summed into a value for each well total of 4 wells. Percent rescue and phospho-Tau induction were calculated as follows:
$${{{{{rm{MAP2}}}}}},{{{{{rm{Percent}}}}}},{{{{{rm{rescue}}}}}}=frac{big({{Area}}_{{{experimental}},{{condition}}}-({{mean}}({{Area}}_{Abeta 42{{treated}}}))}{{{mean}}({{Area}}_{{{{{{rm{untreated}}}}}}})-{{mean}}({{Area}}_{Abeta 42{{treated}}})}$$
(1)
$${{{{{rm{Synapsin}}}}}},{{{{{rm{count}}}}}},{{{{{rm{rescue}}}}}}=frac{big({{Count}}_{{{experimental}},{{condition}}}-({{mean}}({{Count}}_{Abeta 42{{treated}}}))}{{{mean}}({{Count}}{,}_{{{untreated}}})-{{mean}}({{Count}}_{Abeta 42{{treated}}})}$$
(2)
$${{{{{rm{p-Tau}}}}}},{{{{{rm{induction}}}}}}=\ frac{{(D{times}A({{pTau}}396-404))}_{{{experimental}},{{condition}}}/(D{times}A{({{total}},{{Tau}})}_{{{experimental}},{{condition}}})big)}{{(D{times}A({{pTau}}396-404))}_{{{untreated}}}/(D{times}A{({{total}},{{Tau}})}_{{{untreated}}})}$$
(3)
Replicates of 4 were averaged and plotted with error bars representing SEM. D × A = total integrated intensity. IC50 curves were fitted utilizing Prism software.
Peggy Sue automated western
Cells were lysed with Pierce IP lysis buffer (ThermoFisher #87787) containing protease and phosphatase inhibitor cocktail and cleared by centrifugation. Lysates concentration was quantified using the Micro BCA Protein Assay Kit (ThermoFisher #23235) and adjusted with lysis buffer. Sample lysates were then combined with a 5X master mix (ProteinSimple) to achieve a final concentration of 1X sample buffer, in the presence of fluorescent standards and 40 mM dithiothreitol (DTT). The mixture was denatured at 95 °C for 5 min, and proteins were separated using the Peggy Sue system (ProteinSimple, San Jose, California, USA) with a 12–230 kDa Separation Module (ProteinSimple #SM-S001), according to the manufacturer’s standard protocol. Phosphorylated cJun was immune-probed with anti-phospho-cJun (Cell Signaling, #2361), while the secondary antibody was from the ProteinSimple kit. Both antibodies were diluted using an antibody diluent (ProteinSimple). GAPDH was used as an internal reference. The digital image was analyzed with Compass for Simple Western software.
Tau fraction western
Sample preparation and fractionation
iPSC-derived neurons cultured in 96-well plates for 3 months were treated with sAβ42s as in the Aβ42 Oligomer Experiments section above. Cells were treated twice per week at media change, after three weeks the cells were lysed in 50 µl/well NP40 buffer (Thermo FNN0021) with protease and phosphatase inhibitors. Three to four wells were pooled as one sample and fractionated. Lysate was spun at 20k × g for 30 min at 4 °C. The supernatant was taken as the soluble fraction. The resulting pellet was resuspended vigorously in RIPA (Thermo 89900) with 1% sarkosyl detergent (Sigma 61743) and protease and phosphatase inhibitors by pipetting through a small aperture until a homogeneous solution was obtained. The solution was spun at 20k × g for 30 min at 4 °C. The supernatant was removed and the remaining sarkosyl insoluble pellet was resuspended in sample buffer 4x LDS (Genscript M00676) with 5% beta-mercaptoethanol before electrophoresis. The soluble fraction was assayed by Micro BCA Protein Assay Kit (ThermoFisher #23235) to determine protein concentration.
Immunoblotting
Equal soluble protein (2 μg/lane) and a proportional amount of the corresponding insoluble sample were loaded into a 4–12% Bis-Tris Gel (Invitrogen WG1402BOX) and separated in MES buffer (Invitrogen NP0002). Protein was blotted by the iBlot2 device and compatible PVDF stacks (Invitrogen IB24001). Membranes were fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 15 min at RT. Membranes were blocked in blocking buffer 10% donkey serum and 5% BSA (Jackson Immunoresearch) in PBS for 1 h RT. Primary antibodies were diluted in blocking buffer 1:1000 and incubated overnight at 4 °C, rocking (Tau HT7 Thermo MN1000, Histone H3 4499S Cell Signaling Technology, 3R Tau 05-803 EMDMillipore, 4R Tau 05-804 EMDMillipore). Secondary antibody (peroxidase-Donkey anti-mouse 715-035-150, peroxidase-Donkey anti-rabbit 711-033-152, Jackson Immunoresearch) was diluted in blocking buffer (1:20,000) and incubated for 1 h RT rocking. Bands were detected by ECL with Supersignal Femto (Thermo 34094) on AzureBiosystems Azure500 chemi detection.
Gyros Aβ42 immunoassay
The supernatant was collected at the end of each experiment using Bravo liquid handler technology prior to cell fixation and used immediately or frozen at −80 °C. Sample supernatants were diluted 1:500 using 0.05% Tween-20 in 1X PBS. Capture antibody Biotinylated mouse anti-β-amyloid antibody 1–16 6E10 (Biolegend) was diluted to a working concentration of 100 μg/ml in 0.05% Tween-20 in 1X PBS. Detection antibody AlexaFluor647 labeled (ThermoFisher, AlexaFluor647 Antibody Labeling Kit) rabbit anti-Beta Amyloid Monoclonal Antibody (ThermoFisher) was diluted to a working concentration of 25 nM in Rexxip F buffer (Gyros Protein Technologies). All samples were quantified against AggreSure Beta-Amyloid (1–42), Human (Anaspec) oligomerized according to Aβ42 Oligomer Generation protocol above. The standard reagent was diluted to 50 nM in 0.05% Tween-20 in 1X PBS and diluted twofold to 3 pM. Capture and detection antibodies, standards, and samples were loaded onto fully-skirted, 96-well polypropylene plates (ThermoFisher) and run on Gyrolab xPand system on a 1000 nL CD. Assay ran according to Gyros manufacturer standard protocol utilized a 3-step ELISA assay system protocol and two wash buffers: 0.05% Tween-20 in 1X PBS wash buffer and pH 11 wash buffer (Gyros Protein Technologies). Gyros version 6.4 software (Gyros Protein Technologies) was used to measure protein concentrations from Gyros immunoassays.
Incucyte time-lapse movie acquisition
Incucyte movies were acquired according to the manufacturer’s instructions. Images were programmed to acquire once every 30 min in the brightfield spectrum, green fluorescence channel (400 ms acquisition time), and red fluorescence channel (800 ms acquisition time). One image per well in a 384-well plate with a ×20 objective.
Neural stem cell growth medium
0.5X DMEM/F12 (ThermoFisher), 0.5X Neurobasal (ThermoFisher), 1X B27 no Vitamin A (ThermoFisher), 1X N2 (ThermoFisher), 20 ng/ml BDNF (Peprotech), 20 ng/ml FGF-basic (Peprotech), 20 ng/ml EGF (Peprotech), 0.5 mM Glutamax (Gibco), 0.11 mM β-Mercaptoethanol (Sigma-Aldrich), 1X Normocin (InvivoGen), 50 U/ml Penicillin-Streptomycin (ThermoFisher).
Astrocyte medium
1X DMEM/F12 (ThermoFisher), 1X N2 (ThermoFisher), 10% FBS (ThermoFisher), 1X Normocin (InvivoGen), 50 U/ml Penicillin-Streptomycin (ThermoFisher).
Neuron differentiation medium
0.5X DMEM/F12 (ThermoFisher), 0.5X Neurobasal (ThermoFisher), 1X B27 with Vitamin A (ThermoFisher), 1X N2 (ThermoFisher), 5 μg/ml Cholesterol (Sigma-Aldrich), 1 mM Creatine (Sigma-Aldrich), 100 μM Ascorbic Acid, 0.5 mM cAMP (Sigma-Aldrich), 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 μg/ml Laminin, 0.5 mM Glutamax (ThermoFisher), 1X Normocin (InvivoGen), 50 U/ml Penicillin-Streptomycin (ThermoFisher).
Neuron maintenance medium
1X BrainPhys Basal (StemCell Technology), 1X B27 with Vitamin A (ThermoFisher), 1X N2 (ThermoFisher), 5 μg/ml Cholesterol (Sigma-Aldrich), 1 mM Creatine (Sigma-Aldrich), 10 nM β-estradiol, 200 nM Ascorbic Acid, 1 mM cAMP (Sigma-Aldrich), 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 μg/ml Laminin, 0.5 mM Glutamax (ThermoFisher), 1 ng/ml TGF-β1 (Peprotech), 1X Normocin (InvivoGen), 50 U/ml Penicillin-Streptomycin (ThermoFisher).
Microglia media
BrainPhys neuronal media (Stem Cell Technologies) supplemented with 1X B27 with vitamin A (ThermoFisher), 1X N2 Plus media supplement (R&D Systems), 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 mM creatine (Sigma-Aldrich), 200 nM L-ascorbic acid (Sigma-Aldrich), 1 μg/ml mouse laminin (ThermoFisher), 0.5 mM glutamax (ThermoFisher), 0.5X penicillin-streptomycin (ThermoFisher), 1X Normocin (Invivogen), 5 ng/ml TGF-b (Peprotech), 100 ng/ml human IL-34 (Peprotech), 1.5 μg/ml cholesterol (Sigma-Aldrich), 1 ng/ml gondoic acid (Cayman Chemicals), 100 ng/ml oleic acid (Cayman Chemicals), 460 μM Thioglycerol (Sigma-Aldrich), 1X Insulin-Transferrin-Selenium (ThermoFisher), 25 ng/ml rhM-CSF (Peprotech), and 5.4 μg/ml Human Insulin Solution (Sigma).
Statistical analysis summary
Image analysis was performed using supplied InCell6000 analysis software, InCell Investigator. The software was utilized to segment cellular regions of interest such as dendrites, cell bodies, and axons. During image acquisition, 9 images are collected per well, which are then summed into a value for each well total of 4 wells. Percent rescue and phospho-Tau induction were calculated as follows:
$${{{{{rm{MAP2}}}}}},{{{{{rm{Percent}}}}}},{{{{{rm{rescue}}}}}}=frac{big({{Area}}_{{{experimental}},{{condition}}}-({{mean}}({{Area}}_{Abeta 42{{treated}}}))}{{{mean}}({{Area}}{,}_{{{untreated}}})-{{mean}}({{Area}}_{Abeta 42{{treated}}})}quad(1)$$
$${{{{{rm{Synapsin}}}}}},{{{{{rm{count}}}}}},{{{{{rm{rescue}}}}}}=frac{big({{Count}}_{{{experimental}},{{condition}}}-({{mean}}({{Count}}_{Abeta 42{{treated}}}))}{{{mean}}({{Count}}{,}_{{{untreated}}})-{{mean}}({{Count}}_{Abeta 42{{treated}}})}quad(2)$$
$${{{{{rm{p-Tau}}}}}},{{{{{rm{induction}}}}}}=\ frac{{(D{times}A({{pTau}}396-404))}_{{{experimental}},{{condition}}}/(D{times}A{({{total}},{{Tau}})}_{{{experimental}},{{condition}}})big)}{{(D{times}A({{pTau}}396-404))}_{{{untreated}}}/(D{times}A{({{total}},{{Tau}})}_{{{untreated}}})}quad(3)$$
$$Z,{{{{{rm{factor}}}}}}=1-frac{3(({{Stdev}}_{{{noAbeta}}})+({{Stdev}}_{Abeta 42{{treated}}}))}{({{Mean}}{,}_{{{noAbeta}}})-({{Mean}}_{Abeta 42{{treated}}})}$$
(4)
D × A = total integrated intensity. All statistical analyses were performed in Prism software using a two-way ANOVA followed by program-recommended post hoc test for each experiment. Each experiment has N at least four wells; each well with 1000+ neurons as a sum or average of one well quantified. Data in graphs are expressed as mean values ± s.e.m. Error bars represent s.e.m.
Statistics and reproducibility
All graphs shown are taken from a representative experiment. All experiments shown were repeated independently at least three times with similar results.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
