a cell-based assay to assess the tau protein uptake by microglia

A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

On the first day of the experiment prepare the BV-2 cells by first washing them with 1x PBS in the flask in which they have been cultured. Then, incubate them with 0.05% trypsin EDTA at 37 degrees Celsius and 5% CO2 until they detach from the flask. Resuspend the cells in culture medium by pipetting up and down 3 to 5 times.
Count cells and prepare a cell suspension with a final concentration of 100,000 cells per milliliter in culture medium containing 200 micrograms per milliliter heparin. Add 250 microliters of this cell suspension per well in a 96-well tissue culture flat-bottom plate. Incubate the plate at 37 degrees Celsius with 5% CO2 overnight.
After thawing previously prepared pH dye-Tau aggregates on ice, dilute them in 65 microliters per condition of serum-free medium to make a 500-nanomolar solution. Dilute antibodies in 65 microliters of serum-free medium to achieve double the desired concentration. Mix pH dye-Tau aggregates and antibodies in a 96-well U-bottom plate. Seal this dilution plate and incubate overnight at 37 degrees Celsius.
On the following day, remove the medium from the 96-well plate with BV-2 cells. Wash the cells in each well with 100 microliters of room temperature 1x PBS. Remove PBS from the BV-2 cell plate. Use a multichannel pipette to transfer 125 microliters of immunocomplexes from the dilution plate to each well of a 96-well BV-2 cell plate, and incubate for two hours at 37 degrees Celsius with 5% CO2.
After the incubation, remove the immunocomplexes from the cells and wash the cells in each well with 100 microliters of room temperature 1x PBS. After removing the 1x PBS, add 50 microliters of 0.25% trypsin-EDTA, and incubate for 20 minutes at 37 degrees Celsius with 5% CO2. After that, add 200 microliters of culturing medium and resuspend the well by pipetting up and down to detach the cells.
Transfer the cells to a 96-well U-bottom plate and centrifuge it at 400 times g for 5 minutes at 4 degrees Celsius. Place the plate on ice and remove the medium. Add 150 microliters of ice-cold 1x PBS, and centrifuge again at 400 g for 5 minutes at 4 degrees Celsius. Place the plate on ice and wash again by removing previously added 1x PBS and adding 150 microliters of ice-cold PBS per well. Centrifuge again under the same conditions.
Place the plate on ice and wash the cells by removing the previously added PBS, and adding 150 microliters FACS buffer per well. Centrifuge again at 400 g for 5 minutes at 4 degrees Celsius. Place the plate on ice, and remove the previously added FACS buffer, and resuspend the cells in 200 microliters FACS buffer per well. Immediately proceed to analyze by FACS to acquire 20,000 events in the live-cell gate.

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1. BV-2 Microglia 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); such viruses are known for their&#160;in vitro&#160;transforming ability and&#160;in vivo&#160;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&#160;cells/mL.</li>
	<li>Maintain cultures in a humidified atmosphere of 5% CO2&#160;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&#160;et al.&#160;with the difference that no Thioflavin T (ThT) was added to the reaction buffer. Aggregated samples were collected in 1.5 mL centrifuge tubes. The 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&#160;g&#160;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).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The 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 them on ice to avoid overheating.
	<ol>
		<li>Use an amplitude of 65% (with a 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 referred 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&#160;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).&#160;&#160;&#160;&#160; 
		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&#160;until the cells detach from the flask (approximately 5 min).&#160;&#160; 
		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&#160;cells/mL in culture medium containing 200 &#956;g/mL heparin.</li>
		<li>Plate 250 &#181;L of cell suspension (2.5 x 104&#160;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 at a concentration double the final. Mix pH dye-tau aggregates and antibodies in a 96-well u-bottom plate. The 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 overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Day 2 &#8211; Immunocomplexes Uptake
	<ol>
		<li>Remove the 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&#160;g&#160;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&#160;g&#160;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&#160;g&#160;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&#160;events in the live cells gate (see step 4.1).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 200mM</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&#160;</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 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&#160;</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 0.5M, pH8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: De Marco, D. et al., <a href="https://app.jove.com/v/58576">Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia.</a>&#160;J. Vis. Exp. (2018)
This video showcases a cell-based assay quantifying Tau uptake by murine microglia using pH-sensitive fluorescent-labeled Tau aggregates, analyzed via fluorescence-activated cell sorting for a quantitative assessment.

1. BV-2 Microglia Cells Culture
NOTE: Handle BV-2 cells under Biosafety Level 2 containment. The BV-2 cell line produces an enveloped recombinant ...

Begin with a flat-bottom multi-well plate containing adhered murine microglia &#8212; phagocytic cells in the brain.
Introduce a serum-free medium containing immunocomplexes. These complexes consist of antibodies attached to phosphorylated Tau protein aggregates, which are formed during certain neurodegenerative diseases.
The Tau aggregates are labeled with a pH-sensitive green fluorescent dye.
During incubation, these immunocomplexes interact with the Fc receptors of microglial cells, facilitating their engulfment.
This immunocomplex-containing endosome fuses with a lysosome, forming an endolysosome. The acidic environment of the endolysosome causes the dye molecules to fluoresce.
Wash to remove the non-internalized immunocomplexes.
Treat with trypsin-EDTA solution to detach the cells.
Transfer the cell suspension to a U-bottom plate positioned over ice to stabilize the endolysosome. Pellet the cells and discard the supernatant.
Wash and resuspend the cells with FACS buffer.
Using FACS, identify the single live cells and quantify the green fluorescence intensity to assess Tau uptake by microglia.

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视频: 一种基于细胞的测定法，用于评估小胶质细胞对 tau 蛋白的摄取 - 实验

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Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

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We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

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A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

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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.

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A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

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A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia