Name: analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry
Uploaded: 2017-06-22T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

Dette arbejde blev støttet af tilskud fra Agence National pour la Recherche (ANR-12-MALZ-0003-02-P2X7RAD), Association France Alzheimer og Bpifrance. Vores laboratorium støttes også af Inserm, CNRS, Université Pierre et Marie-Curie og programmet &quot;Investissements d&#39;avenir&quot; ANR-10-IAIHU-06 (IHU-A-ICM). Vi vil gerne takke hjælpen fra CELIS cellekultur kernen facilitet.

Alle metoder, der er beskrevet her, er godkendt af Instituttet for Dyrpleje og Brug ved ICM-instituttet og af Darwin Fransk Etisk Dyreudvalg og er omfattet af protokol 01407.02. 1. Fremstilling <ol><li> Forbered fordøjelsesscocktailen i et 1,5 ml rør ved at kombinere følgende for hver mus: 1 ml phosphatbufferet saltvand (PBS); 123 μL fordøjelsesenzym (se tabel over materialer) ved 13 wunsch / ml (stamopløsning), slutkoncentration 1,6 wunsch / ml; Og 5 μl DNase I (se tabel over mater...

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Det har vist sig, at microglia og MDM har forskellige funktioner og fænotyper i CNS, og identifikation og analyse af disse makrofag subpopulationer er derfor essentielle for bedre at forstå neurologiske sygdomme 9 , 18 , 25 . Flowcytometrianalyse ved anvendelse af to markører (CD11b og CD45) muliggør sondringen mellem hver subpopulation ( figur 3C ). Denne strategi blev tidligere valideret ved ...

Mikroglia er de parenkymvæv-residente makrofager i centralnervesystemet (CNS). De spiller to vigtige funktionelle roller: immunforsvar og vedligeholdelse af CNS homeostasen. I modsætning til MDM, der fornyes kontinuerligt fra de hæmatopoietiske stamceller i knoglemarven, adskiller mikrogialcellerne sig fra primitive hæmatopoietiske stamceller, der stammer fra æggeblommehalsen (YS), der koloniserede hjernen under embryonisk udvikling 1 , 2 , 3 . I gnavere spiller transkriptionsfaktoren Myb en afgørende rolle i udviklingen af ​​alle knoglemarv-afledte monocytter og makrofager, men for YS-afledt microglia er denne faktor dispensabel, og differentiering forbliver afhængig af transkriptionsfaktoren PU.14. I det sunde CNS er microglia dynamiske celler, der konstant prøver deres miljø, scaNning og opmåling til invaderende patogener eller vævsskade 5 . Påvisningen af ​​sådanne signaler initierer en vej for at løse skaden. Mikrogliaen skifter hurtigt fra en forgrenet morfologi til en amoeboid, som efterfølges af fagocytose og frigivelse af forskellige mediatorer, såsom pro- eller antiinflammatoriske cytokiner. Afhængigt af deres mikro-miljø kan således aktiveret microglia erhverve et spektrum af forskellige primertilstande 6 . Mikroglia påvirker dybt udviklingen og udviklingen af ​​mange neurologiske lidelser. I gnavermodellerne af Alzheimers sygdom (AD) 7 , Amyotrophic Lateral Sclerosis (ALS) 8 , Multiple Sclerose (MS) 9 eller Parkinsons sygdom (PD) 10 , er microgliane vist at spille en dobbelt rolle, hvilket enten inducerer skadelig neurotoksicitet eller handler På en neuroprotektiv måde, som er afhængig oN den specifikke sygdom, sygdomsfasen og om sygdommen var påvirket af det systemiske immunrum 7 , 8 , 9 , 10 , 11 . De fleste af de CNS-læsioner, der observeres i ovennævnte sygdomme, indeholder en heterogen population af myeloidceller, herunder ikke kun parenkymisk mikroglia, men også perivaskulære og meningeal-makrofager såvel som CNS-infiltrerende MDM. Disse celletyper kan differentielt bidrage til de patofysiologiske mekanismer relateret til skade og reparation 7 , 12 , 13 , 14 , 15 . Den nuværende udfordring for forskere, der studerer disse sygdomsmodeller, er at fastslå om de perifere monocytter og makrofager infiltrerer CNS og i så fald at distancereIndusish den residente microglia fra disse celler. Faktisk er mikrogialcellerne meget plastiske; Når de aktiveres, gentager microglia markørerne, der normalt udtrykkes af perifere monocytter og makrofager. Spørgsmålet er derfor baseret på at identificere markører, der kan skelne den residente mikroglia fra de infiltrerende monocytter og makrofager. Diskrimineringen af ​​disse populationer på hjerneskiver ved immunohistologiske anvendelser er begrænset på grund af manglen på specifikke antistoffer. Flowcytometrianalyse er imidlertid en effektiv teknik til at vurdere ekspressionen af ​​adskillige markører og at skelne cellepopulationer (for eksempel lymfocytter, makrofager / MDM CD11b + CD45 høj og microglia CD11b + CD45 med ) såvel som cellepopulationer 16 , 17 , 18 . Denne protokol beskriver procedurerne for isolering afMononukleære celler fra muse-CNS i neurologiske sygdomsmodeller ved anvendelse af en optimeret enzymatisk vævsdissociation og en densitetsgradientcentrifugering; Såvel som en metode til differentiering af mikroglia- og MDM-populationerne i CNS ved anvendelse af flowcytometri. En anden fremgangsmåde er at eliminere myelin og rense cellerne ved anvendelse af magnetiske perler konjugeret til specifikke antistoffer 19 , 20 , 21 . Myelinfjernelse ved anvendelse af anti-myelinmagnetiske perler er dyrere og påvirker levedygtigheden og udbyttet af isolerede celler 22 . Dette trin og den følgende immunomagnetiske adskillelse af microglia begrænser yderligere undersøgelser af specifikke immuncellepopulationer 21 , 22 . Disse procedurer giver en nem måde at studere makrofag subpopulations på i sygdomsudvikling, ogAt bestemme lægemiddelvirkningerne eller genmodifikationerne på makrofagfænotyper og aktiveringstilstande.

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5-month-old Mice</td>
			<td style="width:223px;">Janvier</td>
			<td style="width:119px;">C57BL/6J</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Liberase TL Research Grade</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">5401020001</td>
			<td>Digestion enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Percoll</td>
			<td style="width:223px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0891-01&#160;</td>
			<td>Density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell Strainer size 70 &#181;m Nylon</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">731751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Venofix 25G</td>
			<td style="width:223px;">BRAUN</td>
			<td style="width:119px;">4056370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Piston syringe 10 mL</td>
			<td style="width:223px;">Terumo</td>
			<td style="width:119px;">SS+10ES1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Pasteur pipette 230 mm</td>
			<td style="width:223px;">Dustcher</td>
			<td style="width:119px;">20420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">1,5 mL&#160; tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">0030 123.328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">15 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">50 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5 mL polystyrene round bottom tube</td>
			<td style="width:223px;">BD Falcon</td>
			<td style="width:119px;">352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (1X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (10X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14200-067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fetal bovine serum</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Bovine Serum Albumin solution 30%</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Paraformaldehyde 32% Solution</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-S</td>
			<td>Caution -Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Saponin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Sodium Azide</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-4031&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat anti-mouse CD45 (clone 30-F11)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">563890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat IgG2b, &#954; Isotype Control RUO</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">562603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rabbit anti-mouse TMEM119 (clone28-3)</td>
			<td style="width:223px;">Abcam</td>
			<td style="width:119px;">ab209064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">AlexaFluor 647 Donkey anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">14-0161</td>
			<td>Mouse Fc Block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fixable Dead Cell Stain Kits</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:119px;">L34969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CCR2 APC-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5538A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2B APC-conjugated Isotype Control</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC013A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CX3CR1 PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5825P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Goat IgG PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC108P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Centrifuge</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">5804R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell analyzer</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">BD FACSVERSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Data Analysis Software</td>
			<td style="width:223px;">FlowJo LLC</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fine scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14090-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Standard Pattern Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11000-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mayo Scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14010-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Dumont #5 Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry

Talrige undersøgelser har vist rollen som immunceller, især makrofager, i sygdomme i centralnervesystemet (CNS). Der er to hovedmakrofagpopulationer i CNS: (i) microgliaen, som er de residente makrofager i CNS og er afledt af æggeblomme-progenitorer under embryogenese, og (ii) de monocyt-afledte makrofager (MDM), som kan infiltrere CNS under sygdom og er afledt af knoglemarvsprogere. Rollerne i hver makrofag subpopulation varierer afhængigt af den patologi, der undersøges. Desuden er der ingen konsensus om de histologiske markører eller de kendetegnende kriterier, der anvendes til disse makrofag subpopulationer. Analysen af ​​ekspressionsprofilerne af CD11b- og CD45-markørerne ved hjælp af flowcytometri tillader os imidlertid at skelne microglia (CD11b + CD45 med ) fra MDM (CD11b + CD45 høj ). I denne protokol viser vi, at densitetsgradientcentrifugeringenOg flowcytometrianalysen kan anvendes til at karakterisere disse CNS-makrofag-subpopulationer og at studere flere markører af interesse udtrykt af disse celler, som vi for nylig offentliggjorde. Således kan denne teknik fremme vores forståelse af makrofagens rolle i musemodeller af neurologiske sygdomme og kan også bruges til at evaluere lægemiddelvirkninger på disse celler.

Efter densitetsgradientcentrifugering og antistoffarvning blev cellerne erhvervet på et flowcytometer og analyseret under anvendelse af en morfologisk gatingstrategi som følger. En første port blev defineret i punktprotot Forward-Spreaded Area (FSC-A) versus Forward-Spreaded-Height (FSC-H) for at diskriminere enkeltceller fra dubletter ( figur 3A ). Enkeltcellerne blev dernæst gated på DSC-A versus Side-Spreaded Area (SSC-A) punkterne ...

Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Denne protokol tilvejebringer en analyse af makrofag subpopulationerne i det voksne mus-centralnervesystem ved hjælp af flowcytometri og er nyttig til undersøgelsen af ​​flere markører udtrykt af disse celler.

Analyse af Microglia og Monocyt-afledte Makrofager fra Centralnervesystemet ved Flowcytometri

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Research

JoVE Journal

Immunology and Infection

Culturing Microglia from the Neonatal and Adult Central Nervous System

Dyrkning Mikroglia fra Neonatal og Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Dyrkning af makrofag kolonistimulerende faktor differentierede humane monocytafledte makrofager

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Karakterisering af Humane monocytafledte dendritiske celler fra Imaging flowcytometri: en sammenligning mellem to Monocyt Isolation protokoller

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Fænotypisk karakterisering af makrofager fra rottenyre ved flowcytometri

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

Kvalitativ og kvantitativ analyse af den immun Synapse i det menneskelige System, ved hjælp af Imaging flowcytometri

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Isolation, karakterisering og rensning af makrofager fra væv ramt af fedme-relaterede Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Karakterisering af humane monocyt delmængder af fuldblod Flow flowcytometri analyse

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Flow cytometrisk analyse af lymfocyt infiltration i centralnervesystemet under eksperimentel autoimmun encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

Isolering af centralnervesystemvæv og tilhørende meninges til downstream-analyse af immunceller

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...