We would like to thank Alberto Carpinteiro Soares for his valuable technical assistance.

1. BV-2 Cells Culture
NOTE: Handle BV-2 cells under Biosafety Level 2 containment. The BV-2 cell line produces an enveloped recombinant ecotropic retrovirus (capable of infecting murine cells only)12; such viruses are known for their in vitro transforming ability and in vivo tumorigenic potential.
<ol>
	<li>Culture BV-2 cells in high glucose Dulbecco&#39;s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 &#181;g/mL streptomycin and 2 mM L-Glutamine (referred to as culturing medium from now on) by seeding cells at 4 x 104 cells/mL.</li>
	<li>Maintain cultures in a humidified atmosphere of 5% CO2 at 37 &#176;C. 
	NOTE: the cells grow loosely attached and in suspension.</li>
</ol>
2. Label Recombinant Tau Aggregates with pH-sensitive Fluorescent Dye
NOTE: Tau aggregates were prepared as described in Apetri et al.13 with the difference that no Thioflavin T (ThT) was added to the reaction buffer. Aggregated samples were collected in 1.5 mL centrifuge tubes. Final fluorescence signal was checked by mixing 118 &#181;L of the pool sample with 12 &#181;L of a 50 &#181;M ThT solution. Aggregates were separated by centrifuging the aggregation reaction mixture at 20,000 x g for 1 h at 4 &#176;C. The supernatant was analyzed by SEC-MALS to confirm that all the monomeric tau was converted into aggregates. Pellets (tau aggregates) were snap frozen and stored in a freezer at -80 &#176;C.
<ol>
	<li>Resuspend tau aggregates in 0.1 M sodium bicarbonate buffer (NaHCO3) at pH 8.5 to a concentration of 1 mg/mL (~ 20 &#181;M). 
	NOTE: Concentration of tau aggregates is based on the initial monomers concentration as assessed by the absorption of tau monomers at 280 nm using an extinction coefficient of 0.31 mLmg-1cm-1.</li>
	<li>Sonicate the resuspended aggregates using a probe sonicator while keeping on ice to avoid over-heating.
	<ol>
		<li>Use an amplitude of 65% (with sonicator of power 250 W).</li>
		<li>Perform 8 pulses of 3 s with pauses of 15 s between pulses to avoid overheating.</li>
	</ol>
	</li>
	<li>Prepare an 8.9 mM stock solution of pH-sensitive&#160;dye (henceforth reference to as pH dye) in dimethyl sulfoxide (DMSO) following manufacturer&#39;s instruction. 
	Note: Always prepare a fresh solution and use it only on the day it is prepared.</li>
	<li>Add 10 moles of dye per mole of protein to a final dye concentration of 0.2 mM.</li>
	<li>Mix by gently pipetting up and down.</li>
	<li>Incubate the reaction mixture for 45&#8211;60 min at room temperature, protected from light.</li>
	<li>In the meantime, assemble a cross-linked dextran gel desalting column following manufacturer&#39;s instructions.</li>
	<li>Equilibrate the column with 25 mL of 0.1 M NaHCO3&#160;buffer&#160;pH 8.5 containing&#160;3% DMSO. Discard the flow through.</li>
	<li>Add the product of the tau aggregate labeling reaction to the column in a total volume of 2.5 mL. If the sample is less than 2.5 mL, add buffer until a total volume of 2.5 mL is achieved.</li>
	<li>Let the sample enter the packed gel completely, discard the flow-through.</li>
	<li>Elute with 3.5 mL of 0.1 M NaHCO3 buffer pH 8.5 containing 3% DMSO&#160;and collect the eluate in 4 equivalent fractions in 2 mL tubes.</li>
	<li>Determine protein concentrations of the 4 fractions by bicinchoninic acid (BCA) assay.</li>
	<li>Store the labeled protein in a -20 &#176;C freezer.</li>
</ol>
3. Uptake Assay with Fluorescence-activated Cell Sorting (FACS) Read-out
<ol>
	<li>Day 1 &#8211; Seed the Cells
	<ol>
		<li>Wash BV-2 cells in the flask by removing culturing medium and adding 1x&#160;phosphate-buffered saline (PBS). 
		NOTE: Washing volume will vary based on the size of the cell flask used. For example, for a T175 flask, wash with 10 mL of 1x PBS.</li>
		<li>Remove PBS from the flask and detach cells by incubating with trypsin-ethylenediaminetetraacetic acid (EDTA) 0.05% at 37 &#176;C and 5% CO2 until the cells detach from the flask (approximately 5 min). 
		NOTE: Volume of trypsin-EDTA 0.05% depends on the size of the cell flask used. For example, for a T175 flask, use 2 mL of trypsin-EDTA 0.05%.</li>
		<li>Resuspend cells in culturing medium by pipetting up and down three to five times. 
		Note:&#160;Volume of culturing medium varies based on the size of the cell flask used and thus total number of cells in the flask. For example, for a T175 flask, use 8 mL of culturing medium.</li>
		<li>Count cells and create a cell suspension with&#160; a final concentration of 1 x 105 cells/mL in culture medium containing 200 &#956;g/mL heparin.</li>
		<li>Plate 250 &#181;L of cell suspension (2.5 x 104 cells) per well in a 96-well tissue culture flat bottom plate.</li>
		<li>Incubate plate overnight at 37 &#176;C and&#160;5% CO2.</li>
	</ol>
	</li>
	<li>Day 1 &#8211; Prepare Immunocomplexes
	<ol>
		<li>Thaw pH dye-tau on ice.</li>
		<li>Prepare 65 &#956;l per condition of a 500 nM solution of pH dye-tau aggregates in serum-free medium (SFM) (high glucose DMEM supplemented with 100 U/mL penicillin, 100 &#181;g/mL streptomycin and 200 &#181;g/mL of heparin).</li>
		<li>Prepare antibody dilutions in 65 &#181;l of SFM and at a concentration double the final one. Mix pH dye-tau aggregates and antibodies in a 96-well u-bottom plate. Final volume per condition is now 130 &#181;l and the pH dye-tau aggregates concentration is 250 nM.&#160;Seal the dilution plate and incubate over night at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Day 2 &#8211; Immunocomplexes Uptake
	<ol>
		<li>Remove culturing medium from BV-2 cells. Wash cells once with 100 &#181;L room temperature 1x PBS.</li>
		<li>Transfer 125 &#181;L of immunocomplexes to the cells using a multichannel pipette. Incubate the cells with the immunocomplexes for 2 h&#160;at 37 &#176;C and&#160;5% CO2.</li>
		<li>Remove the incubation medium from the cells and discard it. Wash cells once with 100 &#181;L room temperature 1x PBS.</li>
		<li>Remove 1x PBS and treat cells with 50 &#181;L trypsin-EDTA 0.25% for 20 min at 37 &#176;C and&#160;5% CO2. </li>
		<li>Add 200 &#181;L of culturing medium and resuspend well by pipetting up and down to detach the cells. Transfer cells to a 96-well U-bottom plate. Centrifuge plate at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Put the plate on ice, remove culturing medium and wash cells twice by resuspending the cell pellets in 150 &#181;L ice cold 1x PBS. Centrifuge plate at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Put cells on ice, remove added 1x PBS and wash them by resuspending cells pellets in 150 &#181;L cold FACS buffer (1x PBS, 0.5% bovine serum albumin (BSA), 2 mM EDTA). Centrifuge plate at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Put cells on ice, remove added FACS buffer and resuspend cells&#160;in 200 &#181;L cold FACS buffer.</li>
		<li>Analyze samples immediately by FACS acquiring 2 x 104 events in the live cells gate (see step 4.1).</li>
	</ol>
	</li>
</ol>
4. FACS Analysis
NOTE: Refer to Figure 1 for the gating strategy.
<ol>
	<li>Using the forward scatter area (FSC-A) versus side scatter area (SSC-A) density plot, gate on live cells by excluding events with lower forward scatter levels (i.e., debris and dead cells).</li>
	<li>Within the live cell population, use FSC-A versus forward scatter height (FSC-H) to exclude cell doublets and aggregates. This is the singlet gate.</li>
	<li>Using the events in the singlet gate, generate a pH dye single parameter histogram.</li>
	<li>Determine mean fluorescence intensity. Determine percentage of pH dye-tau positive cells by excluding negative cells as determined using BV-2 only control.</li>
</ol>
5. Immunocomplexes Uptake with Microscopy Read-out
<ol>
	<li>Day 1 &#8211; Seed the Cells
	<ol>
		<li>Prepare BV-2 cells as described in steps 3.1.1, 3.1.2 and 3.1.3.</li>
		<li>Count cells and resuspend them&#160;in culturing medium to a final concentration of 104 cells/mL.</li>
		<li>Plate 150 &#181;L of cell suspension (1.5&#160;x 103) per well in a poly-D-Lysine coated 96-well black plate with clear flat bottom.</li>
		<li>Incubate plate&#160;at 37 &#176;C and&#160;5% CO2 for 48 h.</li>
	</ol>
	</li>
	<li>Day 3 &#8211; Prepare Immunocomplexes 
	NOTE: Mild sonication of labelled tau aggregates prior to incubation with antibody, was performed to improve microscopy results.
	<ol>
		<li>Thaw pH dye-tau on ice and sonicate using a probe sonicator while keeping on ice. Use an amplitude of 15% (sonicator power of 250 W).Perform 30 pulses of 2 s and wait 20 s between pulses.</li>
		<li>Prepare 65 &#956;l per condition of a 500 nM solution of pH dye-tau aggregates in SFM.</li>
		<li>Dilute antibodies in 65 &#181;l of SFM to a concentration double the final one. Mix pH dye-tau aggregates and antibodies in a 96-well u-bottom plate. Final volume per condition is now 130 &#181;l and the pH dye-tau aggregates concentration is 250 nM. Seal the dilution plate and incubate over night at 37 &#176;C.</li>
		<li>Remove medium from cell plate and replace with 150 &#181;L of culturing medium supplemented with 200 &#181;g/mL heparin. Incubate plate overnight at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Day 4 &#8211; Immunocomplexes Uptake
	<ol>
		<li>Remove culturing medium from the BV-2 cells. Transfer 125 &#181;L of immunocomplexes to the cells using a multichannel pipette.</li>
		<li>Incubate the cells with the immunocomplexes for 1 h and 45 min at 37 &#176;C with 5% CO2. 
		Stain cell nuclei with DNA specific dye and acidic organelles with a probe which selectively stains low pH cellular compartments. Incubate the cells 15 min at 37 &#176;C with 5% CO2. 
		NOTE: Dilute the dyes in SFM.</li>
		<li>Perform live-cell imaging using a high content screening confocal system. Set temperature&#160;to 37 &#176;C and 5% CO2. For high quality images, use a 63X water immersion objective and acquire 0.5 &#181;m planes (20 per Z-stack) per imaged field.</li>
	</ol>
	</li>
</ol>

<ol><li>Hefti, F., Goure, W. F., Jerecic, J., Iverson, K. S., Walicke, P. A., Krafft, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+case+for+soluble+A%CE%B2+oligomers+as+a+drug+target+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Trends+in+Pharmacological+Sciences%22%5BJournal%5D)">The case for soluble Aβ oligomers as a drug target in Alzheimer's disease.</a> Trends in Pharmacological Sciences. 34 (5), 261-266 (2013).</li>
<li>Castillo-Carranza, D. L., Lasagna-Reeves, C. A., Kayed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tau+aggregates+as+immunotherapeutic+targets%5BTitle%5D)+AND+%22Frontiers+in+bioscience+%28Scholar+edition%29%22%5BJournal%5D)">Tau aggregates as immunotherapeutic targets.</a> Frontiers in bioscience (Scholar edition). 5, 426-438 (2013).</li>
<li>Martin, L., Latypova, X., Terro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Post-translational+modifications+of+tau+protein%3A+Implications+for+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Neurochemistry+International%22%5BJournal%5D)">Post-translational modifications of tau protein: Implications for Alzheimer's disease.</a> Neurochemistry International. 58 (4), 458-471 (2011).</li>
<li>Holmes, B. B., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prion-like+properties+of+Tau+protein%3A+The+importance+of+extracellular+Tau+as+a+therapeutic+target%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Prion-like properties of Tau protein: The importance of extracellular Tau as a therapeutic target.</a> Journal of Biological Chemistry. 289 (29), 19855-19861 (2014).</li>
<li>Giacobini, E., Gold, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alzheimer+disease+therapy--moving+from+amyloid-beta+to+tau%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">Alzheimer disease therapy--moving from amyloid-beta to tau.</a> Nature Reviews Neurology. 9 (12), 677-686 (2013).</li>
<li>Asuni, A. A., Boutajangout, A., Quartermain, D., Sigurdsson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunotherapy+targeting+pathological+tau+conformers+in+a+tangle+mouse+model+reduces+brain+pathology+with+associated+functional+improvements%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements.</a> Journal of Neuroscience. 27 (34), 9115-9129 (2007).</li>
<li>Boutajangout, A., Ingadottir, J., Davies, P., Sigurdsson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passive+immunization+targeting+pathological+phospho-tau+protein+in+a+mouse+model+reduces+functional+decline+and+clears+tau+aggregates+from+the+brain%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain.</a> Journal of Neurochemistry. 118 (4), 658-667 (2011).</li>
<li>Chai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passive+immunization+with+anti-Tau+antibodies+in+two+transgenic+models%3A+reduction+of+Tau+pathology+and+delay+of+disease+progression%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Passive immunization with anti-Tau antibodies in two transgenic models: reduction of Tau pathology and delay of disease progression.</a> Journal of Biological Chemistry. 286 (39), 34457-34467 (2011).</li>
<li>Yanamandra, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-tau+antibodies+that+block+tau+aggregate+seeding+in+vitro+markedly+decrease+pathology+and+improve+cognition+in+vivo%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo.</a> Neuron. 80 (2), 402-414 (2013).</li>
<li>Luo, W., Liu, W., Hu, X., Hanna, M., Caravaca, A., Paul, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+internalization+and+degradation+of+pathological+tau+is+enhanced+by+an+anti-tau+monoclonal+antibody%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody.</a> Scientific Reports. 5, 11161(2015).</li>
<li>Funk, K. E., Mirbaha, H., Jiang, H., Holtzman, D. M., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+Therapeutic+Mechanisms+of+Tau+Antibodies%3A+Promoting+Microglial+Clearance+Versus+Blocking+Neuronal+Uptake%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Distinct Therapeutic Mechanisms of Tau Antibodies: Promoting Microglial Clearance Versus Blocking Neuronal Uptake.</a> Journal of Biological Chemistry. 290 (35), 21652-21662 (2015).</li>
<li>Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., Bistoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immortalization+of+murine+microglial+cells+by+a+v-raf%2Fv-myc+carrying+retrovirus%5BTitle%5D)+AND+%22Journal+of+neuroimmunology%22%5BJournal%5D)">Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus.</a> Journal of neuroimmunology. 27 (2-3), 229-237 (1990).</li>
<li>Apetri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+common+antigenic+motif+recognized+by+naturally+occurring+human+VH5-51%2FVL4-1+anti-tau+antibodies+with+distinct+functionalities%5BTitle%5D)+AND+%22Acta+neuropathologica+communications%22%5BJournal%5D)">A common antigenic motif recognized by naturally occurring human VH5-51/VL4-1 anti-tau antibodies with distinct functionalities.</a> Acta neuropathologica communications. 6 (1), 43(2018).</li>
<li>Schneider, A., Mandelkow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tau-based+treatment+strategies+in+neurodegenerative+diseases%5BTitle%5D)+AND+%22Neurotherapeutics%22%5BJournal%5D)">Tau-based treatment strategies in neurodegenerative diseases.</a> Neurotherapeutics. 5 (3), 443-457 (2008).</li>
<li>Congdon, E. E., Gu, J., Sait, H. B., Sigurdsson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+uptake+into+neurons+occurs+primarily+via+clathrin-dependent+Fcgamma+receptor+endocytosis+and+is+a+prerequisite+for+acute+tau+protein+clearance%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Antibody uptake into neurons occurs primarily via clathrin-dependent Fcgamma receptor endocytosis and is a prerequisite for acute tau protein clearance.</a> Journal of Biological Chemistry. 288 (49), 35452-35465 (2013).</li>
<li>Bocchini, V., Mazzolla, R., Barluzzi, R., Blasi, E., Sick, P., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+immortalized+cell+line+expresses+properties+of+activated+microglial+cells%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Research%22%5BJournal%5D)">An immortalized cell line expresses properties of activated microglial cells.</a> Journal of Neuroscience Research. 31 (4), 616-621 (1992).</li>
<li>Koenigsknecht, J. <a target="_blank" href="http://www.jneurosci.org/content/24/44/9838.short">Microglial Phagocytosis of Fibrillar β-Amyloid through a β1 Integrin-Dependent Mechanism.</a> Journal of Neuroscience. 24 (44), 9838-9846 (2004).</li>
<li>Kopec, K. K., Carroll, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alzheimer%27s+%CE%B2-Amyloid+Peptide+1-42+Induces+a+Phagocytic+Response+in+Murine+Microglia%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Alzheimer's β-Amyloid Peptide 1-42 Induces a Phagocytic Response in Murine Microglia.</a> Journal of Neurochemistry. 71 (5), 2123-2131 (2002).</li>
<li>Marsh, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+adaptive+immune+system+restrains+Alzheimer%27s+disease+pathogenesis+by+modulating+microglial+function%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">The adaptive immune system restrains Alzheimer's disease pathogenesis by modulating microglial function.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (9), E1316-E1325 (2016).</li>
</ol>

The authors have nothing to disclose.

Microglia, the resident brain&#39;s immune cells, have been recently identified as important players in antibody-mediated therapeutic approaches for tauopathies10,11. Antibody-mediated tau clearance by microglia, together with blocking of neuronal uptake9, inhibition or destabilization of fibril formation13,14 and clearance of intraneuronal fibrils via the lysosomal pathway15, might all contribute to the anti-tau antibody efficacy observed in mouse model of tauopathy5,6,7,8,9.
We described here a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.
This assay, adapted from Funk et al.11, uses BV-2 cells, which are immortalized murine microglial cells. While they cannot fully be compared to primary microglial cells, they feature many of the characteristics of primary microglia, including the ability of robustly phagocytose both A&#946; and tau fibrils11,16,17,18,19. Moreover, they showed a reproducible behavior in vitro which made them highly suitable for assay development and quantitative studies, which require minimal experimental variability. Beside this, immortalized cell lines allow higher assay throughput and eliminate the need for animal sacrifice compared to the use of primary microglia.
The tau aggregates we used in this assay were obtained using the highly reproducible in vitro aggregation procedure that we recently described13, and show similar morphology to paired helical filaments (PHFs) isolated from brains of AD patients. While we did not observe any unexpected results that might have been caused by tau aggregates adherence to plastic or glass surfaces, the use of stable and well characterized tau aggregates played a crucial role in the reproducibility of this assay.
Another aspect that significantly contributed to assay reproducibility was cell density. The numbers of cells per well described in the protocol represent the optimal cell density in the described conditions.
Differently than what Funk et al.11 described, we labeled tau aggregates with a pH sensitive dye&#160;which significantly increases its fluorescence upon internalization in acidic organelles, thus allowing for intracellular quantification. This, together with trypsin digestion of surface bound immunocomplexes and/or tau, guarantees that fluorescence signal measured by flow cytometry is the result of tau uptake rather than binding to the cellular surface. Moreover, the use of a pH sensitive dye eases detection of internalized tau aggregates in microscopy experiments without the need of digesting surface bound immunocomplexes/tau aggregates which would then requires cell re-plating and recovery.
We also further optimized the microscopy read-out of our assay, compared to what has previously been described11, by using a highly selective dye for acidic organelles&#160;in our microscopy experiments which allowed us not only to confirm antibody-mediated tau uptake, but also localization of tau aggregates in the endolysosomal compartment.
The assay we developed, has optimal specificity which results in a good experimental window allowing a strong separation between positive and negative samples. Interestingly, the assay indirectly detects differences in antibody affinity thus representing a powerful tool to study anti-tau antibody effector functions.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition characterized by the conformational change and self-assembly of amyloid &#946; peptide and tau protein into pathological aggregates. The normal soluble amyloid &#946; peptide is converted into oligomeric and fibrillar amyloid &#946;, while abnormally phosphorylated tau accumulates as oligomers and neurofibrillary tangles1,2. These protein aggregates cause neuronal death leading to memory loss and subsequent progressive cognitive decline. Other factors, including non-productive neuroinflammation and a reduced ability to clear misfolded proteins, may exacerbate and accelerate disease. Currently, intervention strategies against AD provide largely symptomatic relief, but there is no disease-modifying cure or prevention.
Increasing evidence suggests a key role of hyperphosphorylated tau aggregates in the pathology of AD. In its non-pathological state, tau is a natively unfolded protein that binds to microtubules and promotes their assembly into the neuronal cytoskeleton. When tau becomes hyperphosphorylated, it detaches from the cytoskeleton and clusters into tau aggregates in the neuron, which are believed to cause most of the pathology associated with AD3. Aggregated tau starts accumulating first intracellularly, but as disease progresses, it is assumed to be released from affected neurons into the extracellular space, from which it can be taken up by adjacent or synaptically connected healthy neurons in a &#34;prion-like manner&#34;. Once internalized, the tau aggregate induces further tau aggregation via templated conformational change4.
According to this hypothesis, therapies capable of interrupting tau seeding might slow down or reverse the course of tau-mediated neurodegenerative disease. In support of this, mice made susceptible to tauopathy by genetic mutation and passively injected with anti-tau antibodies show reduced tau pathology and improved cognitive function5,6,7,8,9. However, the mechanisms by which therapeutic antibodies reduce pathology still remain elusive.
One of the proposed mechanisms is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia, the brain&#39;s resident immune cells. Recent publications suggest that microglia can efficiently internalize and degrade pathological tau species and this ability is enhanced by anti-tau antibodies via an Fc-dependent mechanism involving Fc receptors expressed on the surface of microglia and receptor mediated phagocytosis10,11. These data identify microglia as potentially important effectors of therapeutic antibodies.
We describe herein a cell-based assay to quantitatively assess tau uptake by microglia. Data generated with this assay can help elucidating the mechanisms of action of anti-tau antibodies thus representing a useful tool to advance anti-tau antibodies to further&#160;steps of their development as potential AD treatment.

<table><tbody><tr><td>BV-2 cells</td><td>ICLC Interlab Cell Line Collection</td><td>ATL03001</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) (1x)</td><td>Gibco</td><td>10010-015</td><td></td></tr><tr><td>Trypsin-EDTA 0.05%</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>DMEM 4.5 g/dL glucose</td><td>Gibco</td><td>41966-029</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10091-148</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine 200 mM</td><td>Lonza</td><td>17-605E</td><td></td></tr><tr><td>EasYFlask</td><td>Nunc</td><td>156499 / 159910</td><td></td></tr><tr><td>pHrodo Green STP ester</td><td>Life Technologies</td><td>P35369&amp;nbsp;</td><td></td></tr><tr><td>Sodium Bicarbonate pH 8.5 100 mM</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-100ml</td><td></td></tr><tr><td>PD10 columns</td><td>GE Healthcare</td><td>17-0851-01</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Greiner CELLSTAR&amp;nbsp;multiwell culture plates</td><td>Greiner</td><td>665180</td><td></td></tr><tr><td>Falcon 96-Well Assay Plates</td><td>Falcon</td><td>353910</td><td></td></tr><tr><td>Heparin</td><td>Sigma&amp;nbsp;</td><td>H3393-50KU</td><td></td></tr><tr><td>Trypsin-EDTA 0.25%</td><td>Sigma</td><td>T4049-100ml</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7030-100G</td><td></td></tr><tr><td>EDTA 0.5 M, pH8</td><td></td><td></td><td></td></tr><tr><td>FACS Canto II</td><td>BD</td><td></td><td></td></tr><tr><td>Hoechst 33342 Solution (20 mM)</td><td>Thermo Fisher Scientific</td><td>62249</td><td></td></tr><tr><td>LysoTracker Deep Red</td><td>Thermo Fisher Scientific</td><td>L12492</td><td></td></tr><tr><td>Opera Phenix</td><td>Perkin Helmer</td><td>HH14000000</td><td></td></tr></tbody></table>

cell-based assay to study antibody-mediated tau clearance by microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Aggregated recombinant tau was covalently labelled with a pH-sensitive green dye. This dye dramatically increases its fluorescence upon its internalization in acidic organelles, thereby allowing for intracellular quantification. Labeled tau aggregates were incubated with anti-tau monoclonal antibodies. In particular, we used a chimeric version (mouse IgG1 Fc region) of CBTAU-28.1. This human antibody binds to the N-terminal insert region of tau and is able to bind in vitro generated tau fibrils13. In this assay, we also tested an affinity-improved version of CBTAU-28.1 &#8211; dmCBTAU-28.1. Fab fragments of CBTAU-28.1, in the parental and high-affinity mutant format, and a mouse IgG1 isotype control were used as controls.
BV-2 cells were incubated with the pre-formed immunocomplexes or aggregated tau alone for two hours&#160;in the presence of heparin to block antibody-independent tau uptake. After incubation, cells were trypsinized to remove the tau bound to the extracellular membrane&#160;and were analyzed for tau uptake by flow cytometry. As we recently described13, we observed that CBTAU-28.1 variants promoted uptake of tau in BV-2 cells in a dose-dependent manner. The uptake was Fc mediated since CBTAU-28.1 Fab fragments did not increase basal tau uptake (Figure 2). Moreover, the high affinity dmCBTAU-28.1 antibody mediated tau uptake into BV-2 cells to a higher extent than the wild-type antibody (Figure 2).
Antibody-mediated tau uptake and localization of tau aggregates in the endolysosomal compartment was confirmed by confocal microscopy (Figure 3) where the acidic cellular compartment was stained using a probe selective for low pH organelles. Intracellular puncta of green pH dye labeled tau aggregates were observed inside the cells that were incubated with CBTAU-28.1. Moreover, intracellular tau aggregates often colocalized with the low pH compartment selective red dye&#160;thus suggesting presence of tau aggregates in the acidic organelles. CBTAU-28.1 Fab fragments did not increase tau uptake again indicating an Fc-receptor mediated internalization mechanism (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58576/58576fig1.jpg" /> 
Figure 1: Gating strategy used in flow cytometry analysis to detect tau internalization by BV-2 cells. Sample data from BV-2 only control (A-C), isotype control (D-F) and dmCBTAU-28.1 (G-I) are shown. BV-2 cell population was gated on a FSC-A vs SSC-A density plot excluding debris and dead cells (A, D, G). BV-2 cells were then further gated on a FSC-A vs FSC-H density plot to exclude cell doublets and aggregates (B, E, H). Single cell gate was used to generate a pH dye (FITC in these representative results) single parameter histogram (C, F, I) and determine geometric mean fluorescence intensity. Alternatively, percentage of pH dye-tau positive cells was calculated excluding negative cells as determined by using BV-2 only control. <a href="https://www.jove.com/files/ftp_upload/58576/58576fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58576/58576fig2.jpg" /> 
Figure 2: CBTAU-28.1 mediates uptake of tau aggregates into microglial BV-2 cells.&#160;Aggregated recombinant tau was covalently labelled with green fluorescence pH-sensitive dye and incubated with a mouse chimeric version of the human anti-tau antibody CBTAU-28.1, its affinity improved format, dmCBTAU-28.1, the corresponding Fab fragments, a mouse IgG1 isotype control antibody or no antibody (tau aggregates alone). Immunocomplexes were subsequently incubated with BV-2 cells for two hours&#160;in the presence of heparin to block antibody-independent tau uptake. Uptake of immunocomplexes was assessed by flow cytometry and expressed as the geometric mean (GM) of fluorescence intensity (A) or percentage of tau positive (tau+) cells (B). Error bars in (A) indicate the standard deviation of two independent experiments, while (B) shows a single experiment. <a href="https://www.jove.com/files/ftp_upload/58576/58576fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58576/58576fig3.jpg" /> 
Figure 3: Tau aggregates are internalized by BV-2 cells and localize in cellular acidic organelles.&#160;Preformed tau-antibody immunocomplexes were incubated with BV-2 cells for two hours&#160;in the presence of heparin to block antibody-independent uptake. After incubation, nuclei were stained with a DNA specific blue dye and the acidic cellular compartment with a low pH compartment selective red dye. Live-cell imaging revealed intracellular puncta of labeled tau aggregates (green) inside the cells that were incubated with CBTAU-28.1 and dmCBTAU-28.1, but not with the isotype control. Moreover, intracellular tau aggregates often colocalized with the red dye (yellow) thus suggesting presence of tau aggregates in the acidic cellular compartment. CBTAU-28.1 Fab fragments did not increase tau uptake indicating an Fc-receptor mediated internalization mechanism. Images represent maximum intensity projections of a 20 planes Z-stack (0.5 &#181;m planes) acquired with a 63X water immersion objective. <a href="https://www.jove.com/files/ftp_upload/58576/58576fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia at JoVE.com

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

cell-based-assay-to-study-antibody-mediated-tau-clearance-by-microglia

Research

JoVE Journal

Neuroscience

8.3K Views.  Janssen Pharmaceutical Company of Johnson & Johnson, Belgium. Een van de voorgestelde mechanismen waarmee therapeutische anti-Tau-antilichamen pathologie bij de ziekte van Alzheimer verminderen, is opname in klaring van pathologische geaggregeerde voormalige Tau door microglia. Hier presenteren we de cel basis test om Tau opname te beoordelen door microglia. Deze test is een nuttig onderzoeksinstrument om het werkingsmechanisme van anti-Tau-antilichamen beter te karakteriseren.Bereid op de eerste dag van het experiment de BV-2 cellen voor door ze...

Video: Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Biochemistry

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

Genetics

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

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