Name: fluorescence-activated cell sorting-radioligand treated tissue (facs-rtt) to determine the cellular origin of radioactive signal
Uploaded: 2021-09-10T14:30:00
Description: Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal at JoVE.com

This work was supported by the Swiss National Science Foundation (grant no. 320030-184713). Authors BBT and KC are supported by the Velux Foundation (project n. 1123). Author ST received support from the Swiss National Science Foundation (Early Post-Doc Mobility Scholarship, no. P2GEP3_191446), the Prof Dr. Max Cloetta Foundation (Clinical Medicine Plus scholarship), and the Jean and Madeleine Vachoux Foundation.

All experimental procedures were conducted in agreement with the Ethics Committee for Human and Animal Experimentation of the Canton of Geneva, the Cantonal Commission for Research Ethics (CCER), and the General Direction Of The Health Of The Canton Of Geneva (Switzerland), respectively. Data are reported following Animal Research: Reporting In-vivo Experiments (ARRIVE) guidelines.
1. SPECT camera preparation and calibration
<ol>
	<li>Turn on the camera, load the operating software ...

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+as+a+central+mechanism+in+Alzheimer's+disease.">Inflammation as a central mechanism in Alzheimer's disease.</a> Alzheimer's &amp; Dementia. 4, 575-590 (2018).</li><li>D'Amelio, M., Puglisi-Allegra, S., Mercuri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dopaminergic+midbrain+in+Alzheimer's+disease:+Translating+basic+science+into+clinical+practice.">The role of dopaminergic midbrain in Alzheimer's disease: Translating basic science into clinical practice.</a> Pharmacological Research. 130, 414-419 (2018).</li><li>D'Amelio, M., Serra, L., Bozzali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventral+tegmental+area+in+prodromal+Alzheimer's+disease:+Bridging+the+gap+between+mice+and+humans.">Ventral tegmental area in prodromal Alzheimer's disease: Bridging the gap between mice and humans.</a> Journal of Alzheimer's Disease: JAD. 63 (1), 181-183 (2018).</li><li>Cohen, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+Alzheimer+rat+with+plaques,+tau+pathology,+behavioral+impairment,+oligomeric+a&#946;,+and+frank+neuronal+loss.">A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric a&#946;, and frank neuronal loss.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (15), 6245-6256 (2013).</li><li>Morrone, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+differences+in+Alzheimer's+disease+pathology+confound+behavioural+rescue+after+amyloid-&#946;+attenuation.">Regional differences in Alzheimer's disease pathology confound behavioural rescue after amyloid-&#946; attenuation.</a> Brain: A Journal of Neurology. 143 (1), 359-373 (2020).</li><li>Berkowitz, L. E., Harvey, R. E., Drake, E., Thompson, S. M., Clark, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+impairment+of+directional+and+spatially+precise+trajectories+by+TgF344-Alzheimer's+disease+rats+in+the+Morris+Water+Task.">Progressive impairment of directional and spatially precise trajectories by TgF344-Alzheimer's disease rats in the Morris Water Task.</a> Scientific Reports. 8 (1), 16153 (2018).</li><li>Koulousakis, P., vanden Hove, D., Visser-Vandewalle, V., Sesia, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+improvements+after+intermittent+deep+brain+stimulation+of+the+nucleus+basalis+of+meynert+in+a+transgenic+rat+model+for+Alzheimer's+disease:+A+preliminary+approach.">Cognitive improvements after intermittent deep brain stimulation of the nucleus basalis of meynert in a transgenic rat model for Alzheimer's disease: A preliminary approach.</a> Journal of Alzheimer's Disease: JAD. 73 (2), 461-466 (2020).</li><li>Tournier, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+reference+learning+deficits+in+absence+of+dysfunctional+working+memory+in+the+TgF344-AD+rat+model+of+Alzheimer's+disease.">Spatial reference learning deficits in absence of dysfunctional working memory in the TgF344-AD rat model of Alzheimer's disease.</a> Genes, Brain, and Behavior. , 12712 (2020).</li><li>Tournier, B. B., Tsartsalis, S., Ceyz&#233;riat, K., Garibotto, V., Millet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+TSPO+signal+and+neuroinflammation+in+Alzheimer's+disease.">In vivo TSPO signal and neuroinflammation in Alzheimer's disease.</a> Cells. 9 (9), (2020).</li><li>Backes, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[11C]raclopride+and+extrastriatal+binding+to+D2/3+receptors.">[11C]raclopride and extrastriatal binding to D2/3 receptors.</a> NeuroImage. 207, 116346 (2020).</li><li>Millet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+dopamine+D(2/3)+receptors+in+rat+brain+using+factor+analysis+corrected+[18F]Fallypride+images.">Quantification of dopamine D(2/3) receptors in rat brain using factor analysis corrected [18F]Fallypride images.</a> NeuroImage. 62 (3), 1455-1468 (2012).</li><li>Tsartsalis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+simplified+reference+tissue+model+for+the+quantification+of+dopamine+D2/3+receptors+with+[18F]Fallypride+images.">A modified simplified reference tissue model for the quantification of dopamine D2/3 receptors with [18F]Fallypride images.</a> Molecular Imaging. 13 (8), (2014).</li><li>Schwarz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescence-activated+cell+sorting+to+examine+cell-type-specific+gene+expression+in+rat+brain+tissue.">Using fluorescence-activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.</a> Journal of Visualized Experiments: JoVE. (99), e52537 (2015).</li><li>Tournier, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+to+reveal+the+cell+origin+of+radioligand+binding.">Fluorescence-activated cell sorting to reveal the cell origin of radioligand binding.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 40 (6), 1242-1255 (2020).</li><li>Tournier, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+TSPO+upregulation+appears+before+microglial+TSPO+in+Alzheimer's+disease.">Astrocytic TSPO upregulation appears before microglial TSPO in Alzheimer's disease.</a> Journal of Alzheimer's Disease: JAD. 77 (3), 1043-1056 (2020).</li></ol>

To our knowledge, this technique was the first to describe an approach that allows a better understanding of in vivo binding alterations of a radiotracer at the cellular level. The protocol describes a multiscale method to quantify radiotracer binding at the cellular level using [125I]CLINDE (TSPO) or [125I]R91150 (5HT2AR) as examples.
This technique is robust and sensitive enough to precisely detect the cellular origin of a wide spectrum of glial cell...

Neurodegenerative diseases, such as Alzheimer&#39;s Disease (AD), are characterized by a neuronal loss associated with increased symptoms. AD, the most common cause of dementia, accounting 60%-70% of cases, affects around 50 million people worldwide1. At a neuropathological level, the two major characteristics of AD are the accumulation of extracellular amyloid-&#946; (A&#946;) plaques and intracellular Tau neurofibrillary tangles. Glial cell alterations have also been associated with AD2 and possible disruption of several neurotransmitter systems3,4.
The TgF344-AD rat line has been modified to model AD by expressing human APP and PS1&#916;E9 transgenes, leading to soluble and insoluble A&#946;-40 and A&#946;-42 expression and amyloid plaque formation5. It also presents the accumulation of hyperphosphorylated forms of the Tau protein leading to tauopathy. From the age of 9-24 months, the rats progressively develop the pathological hallmarks of AD and a cognitive impairment5,6,7,8,9.
Positron Emission Tomography (PET), Single-Photon Emission computed Tomography (SPECT), and autoradiography are techniques based on the emission and quantification of &#947; rays. Radiotracers are quantified either in vivo (PET and SPECT) or ex vivo/in vitro (autoradiography). Those sensitive techniques have contributed to the understanding of mechanisms of several brain diseases, such as AD. Indeed, in terms of neuroinflammation, there are a lot of studies assessing 18 kDa Translocator Protein (TSPO), an in vivo neuroinflammation marker, with radiolabeled tracers such as [11C]-(R)-PK11195 or [11C]PBR28 (for review see10). In addition, alterations of neurotransmitter systems have been studied using radiotracers11,12,13.
However, those techniques do not determine the cellular origin of the radioactive signal. This could hamper the interpretation of the biological underpinnings of the alteration in the binding of a radioligand in PET/SPECT. For instance, in the case of TSPO studies of neuroinflammation, understanding whether the increase or decrease of TSPO is due to astrocytic or microglial changes is of paramount importance. The Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT) technique was developed to get around these problems, allowing the assessment of radioligand binding in every cell type separately and the quantification of the target-protein density per cell. This innovative technique is consequently complementary and highly compatible with PET and SPECT imaging.
Here, this technique was applied along two axes: the study of neuroinflammation using TSPO-specific radioligands and assessing the serotonergic system. On the first axis, the aim was to understand the cellular origin of the TSPO signal in response to an acute inflammatory reaction. Therefore, FACS-RTT was used on the brain tissues of rats after the induction of neuroinflammation via a lipopolysaccharide (LPS) injection and following an in vivo [125I]CLINDE SPECT imaging study. Further, the same imaging and FACS-RTT protocol were applied on 12- and 24-month-old TgF344-AD rats and matching wild-type (WT) rats. The second axis aimed to determine the origin of serotoninergic system alterations in this rat model via ex vivo 5-HT2AR density assessment by cell type.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioVet</td>
			<td>BioVet</td>
			<td></td>
			<td>Software for vitals check</td>
		</tr>
		<tr>
			<td>Bondclone C18 reverse-phase column</td>
			<td>Phenomenex, Schlieren, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Des-Sur</td>
			<td>University Hospital of Geneva</td>
			<td></td>
			<td>Virucide</td>
		</tr>
		<tr>
			<td>Fc Block / anti-CD32</td>
			<td>BD Biosciences</td>
			<td>BDB550270</td>
			<td>Reactivity for rat</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-rat CD90</td>
			<td>Biolegend</td>
			<td>202504</td>
			<td>Reactivity for rat</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>B. Braun</td>
			<td>B01AB01</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Knauer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insyte-W 24 GA 0.75 IN 0.7 x 19 mm</td>
			<td>BD Biosciences</td>
			<td>321312</td>
			<td>24 G catheter</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>ZDG9623</td>
			<td></td>
		</tr>
		<tr>
			<td>Lacryvisc</td>
			<td>Alcon</td>
			<td>2160699</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore soft tape</td>
			<td>3M</td>
			<td>F51DA01</td>
			<td></td>
		</tr>
		<tr>
			<td>MILabs-Uspect II</td>
			<td>MILabs</td>
			<td></td>
			<td>Software for SPECT Camera</td>
		</tr>
		<tr>
			<td>MoFlo Astrios</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>Myelin Removal Beads II</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-733</td>
			<td>Contains beads and myelin removal buffer.</td>
		</tr>
		<tr>
			<td>NaCl 0.9% Sterile solution</td>
			<td>B. Braun</td>
			<td>395202</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td>Contains the enzyme mixes, pipets 1, 2 and 3.</td>
		</tr>
		<tr>
			<td>Nylon Mesh Sheet</td>
			<td>Amazon</td>
			<td>CMN-0074-10YD</td>
			<td>40 inch width, 80 micron size mesh</td>
		</tr>
		<tr>
			<td>Peracetic acid</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>R91150 pr&#233;cursor</td>
			<td>CERMN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak C18 Column</td>
			<td>Waters</td>
			<td></td>
			<td>Concentration column</td>
		</tr>
		<tr>
			<td>Sodium iodide Na125</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tributylin precursor</td>
			<td>CERMN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-SPECT Rec2.38c</td>
			<td>MILabs</td>
			<td>Version Rec2.38c</td>
			<td>Software for SPECT images reconstruction</td>
		</tr>
		<tr>
			<td>USPECT II</td>
			<td>MILabs</td>
			<td></td>
			<td>Spect Camera</td>
		</tr>
		<tr>
			<td>Wizard 3&#34;</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Gamma counter</td>
		</tr>
	</tbody>
</table>

fluorescence-activated cell sorting-radioligand treated tissue (facs-rtt) to determine the cellular origin of radioactive signal

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their alterations are perhaps associated with a pro-inflammatory state. The TgF344-AD rat strain has been designed to express human APP and human PS1&#916;E9 genes, encoding for amyloid proteins A&#946;-40 and A&#946;-42 and displays amyloid pathology and cognitive deficits with aging. The TgF344-AD rat model is used in this study to evaluate the cellular origin of the 18 kDa translocator protein (TSPO, a marker of glial cell activation) binding, and the 5HT2A-receptor (5HT2AR) serotonin receptor levels that are possibly disrupted in AD. The technique presented here is Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT), a quantitative cell-type-specific technique complementary to in vivo PET or SPECT or ex vivo/in vitro autoradiography techniques. It quantifies the same radiolabeled tracer used prior for imaging, using a &#947; counter after cytometry cell sorting. This allows determining the cellular origin of the radiolabeled protein with high cellular specificity and sensitivity. For example, studies with FACS-RTT showed that (i) the increase in TSPO binding was associated with microglia in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation, (ii) an increase in TSPO binding at 12- and 18-months was associated with astrocytes first, and then microglia in the TgF344-AD rats compared to wild type (WT) rats, and (iii) the striatal density of 5HT2AR decreases in astrocytes at 18 months in the same rat AD model. Interestingly, this technique can be extended to virtually all radiotracers.

WT rats experienced in vivo SPECT scan with [125I]CLINDE radiotracer after a unilateral LPS injection (Figure 2). This scan (using summed data from images of 45-60 min post radiotracer injection) showed higher binding of [125I]CLINDE in the site of the LPS injection (Figure 2A) than in the contralateral region of the brain (Figure 2B). The ex vivo samples that underwent FACS-RTT confirmed thos...

Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal at JoVE.com

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer&#39;s Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their ...

This protocol is complementary to in vivo imaging. It allows cell type and cellular level quantification of radiotracers, which is a different scale from the one obtained in vivo. The main advantage of this technique is its sensitivity and the cellular resolution given by the use of radioligands and the cell sorting.Turn on the camera and load the operating software. Click on the Home XYZ stage button to perform homing. Set up the experiment composed of one 10-minute scan acquisition.Set the Step mode to fine and the Acquisition mode to the Listmode to record the entire emission spectrum. Set up the bed and ensure that the warming system, breathing sensor, and anesthesia are functional and secure. Then, place a phantom where the animal's head will be positioned.Set the scan area by sliding the cursors for the three dimensions with the help of the three images in the bottom part of the screen. Ensure that the scan volume of the phantom and the animal is the same. Start the phantom scan for subsequent calibration with the previously established parameters.Ensure to work in an appropriate environment, authorized for experiments involving radioactivity. Incubate 100 micrograms of tributylin precursor in 100 microliters of acetic acid with sodium iodide and five microliters of 37%paracetic acid at 70 degrees Celsius for 20 minutes in a thermocycler positioned in a glove box. Dilute the reaction using 50%acetonitrile in water to reach a volume of 500 microliters.Inject the 500 microliters of the diluted reaction onto a reverse-phase column. Isolate the CLINDE with a linear-gradient HPLC run from 5 to 95%ACN in seven-millimolar phosphoric acid for 10 minutes. Dilute the isolate in water to attain a final volume of 10 milliliters and then inject the diluted reaction onto a concentration column.Elute the CLINDE from the column with 300 microliters of absolute ethanol and then evaporate the ethanol by incubating it in a vacuum centrifuge at room temperature for 40 minutes. Dilute the residue containing the CLINDE in 300 microliters of saline to create a stock solution. After measuring its radioactivity, dilute the stock solution in saline to obtain a solution of 0.037 megabecquerel in 500 microliters.Establish the calibration curves with cold reference compounds and resolve the linear regression equation:Y equal to AX plus B to obtain A and B coefficients. X represents the mass of cold compound, and Y represents the area under the curves. Check the area under the curve of the radioligand, measuring ultraviolet absorbance at 450 nanometers based on the calibration curve and the area under the curve of the radioligand.Estimate the mass and measure the activity of the radioligand. Ensure that the specific activity is greater than 1, 000 gigabecquerel per micromole. Click on the Update image button to update the animal position.Set the scan area by sliding the cursors with the help of the three images in the bottom part of the screen for the three dimensions. Set up the experiment on a 60-minute scan constituting 60 frames of one minute. Reuse all other parameters set up initially.Inject 500 microliters of the radioactive radiotracer and then flush the tube with 300 microliters of sterile 0.9%sodium chloride. Simultaneously, click on Start acquisition to start the scan. Open the scan reconstruction software and then open the dataset, looking for the file name parameters file created in the folder of the scan.Select the isotope of interest. Note that the list mode parameter allows multiple isotope selection at this step. Set the different reconstruction parameters like the voxel size, number of subsets, and iterations as described in the manuscript.Select the output format as NIFTI and then select Start SPECT reconstruction. Use a flat metal spatula and a razor blade to dissect the regions of interest of the brain. Place the tissues in a two-milliliter centrifuge tube and weigh the tissue obtained.Put the samples into a two-milliliter centrifuge tube with one milliliter of calcium-and magnesium-free HBSS and then centrifuge at 300 times G for two minutes at room temperature and remove the supernatant without disturbing the pellet. Add 1, 900 microliters of enzyme mix one and incubate it for 15 minutes at 37 degrees Celsius while agitating the tubes by inversion every five minutes. Add 30 microliters of enzyme mix two.Agitate gently with a 1, 000 microliter pipette back and forth 30 times. Then, incubate the mixture for 15 minutes at 37 degrees Celsius while agitating the tubes by inversion every five minutes. Mix gently back and forth with a 200-microliter pipette and then a 10-microliter pipette before incubating the mixture for 10 minutes at 37 degrees Celsius to dissociate the tissues.Filter the cells with a 70-micrometer cell strainer and add 10 milliliters of calcium-and magnesium-free HBSS. Centrifuge at 300 times G for 10 minutes at room temperature and remove the supernatant without disturbing the pellet. For myelin depletion, resuspend the pellet with 400 microliters of myelin removal buffer and then add 100 microliters of myelin removal beads before incubating for 15 minutes at four degrees Celsius.Add five milliliters of myelin removal buffer and centrifuge 300 times G for 10 minutes at room temperature. Add 500 microliters of myelin removal buffer and place the suspended pellets into the magnetic field column. Wash the column with one milliliter of myelin removal buffer four times.Centrifuge at 300 times G for two minutes at room temperature and remove the supernatant without disturbing the pellet. Vortex briefly to dissociate the cells and add five microliters of Fc Block CD32. Add 100 microliters of the mix of primary antibodies of interest and incubate for 20 minutes at four degrees Celsius.Centrifuge at 350 times G for five minutes at four degrees Celsius and remove the supernatant without disturbing the pellet. Blot the tubes upside down on soft paper. Vortex the tube briefly, then add 100 microliters of the secondary antibody mix and incubate for 15 minutes at four degrees Celsius.Add one milliliter of myelin removal buffer and repeat the centrifugation. Discard the supernatant and blot the tubes upside down on soft paper. Resuspend the cells in 250 microliters of sterile PBS and proceed directly to cell sorting.The in vivo SPECT scan of wild-type rats showed higher binding of CLINDE in the site of the lipopolysaccharide injection than in the contra-lateral region of the brain. The ex vivo samples that underwent fluorescence activated cell sorting radioligand-treated tissue, or FACS-RTT, revealed the presence of a higher number of CLINDE binding sites only in microglia, showing that the cellular origin of the CLINDE signal in the ipsilateral side of the brain was microglia. The increase in translocator protein, or TSPO binding, at 12 months in TgF344-AD rats was restricted to astrocytes.In 24-month-old rats, the increase in TSPO binding was observed due to both astrocytic and microglial alterations. In older TgF344-AD rats, striatal astrocytes displayed a decreased 5HT2AR density when compared with wild-type. A cortical overexpression of TSPO was observed in both astrocytes and microglia of AD subjects compared with age-matched controls when FACS-RTT was performed on human AD postmortem samples.When working with radioactivity, follow the instruction of radio-protection and wear appropriate personal protection equipment. Then, after the procedure, make sure to decontaminate the working environment. Protein quantification and gene expression analysis can easily be done after this procedure on isolated cell populations.

Research

JoVE Journal

Neuroscience

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...