An efficient method to limit microglia-dependent effects in astroglial cultures.

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ContentslistsavailableatSciVerseScienceDirect

Journal

of

Neuroscience

Methods

j o ur n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / j n e u m e t h

Basic

Neuroscience

An

efficient

method

to

limit

microglia-dependent

effects

in

astroglial

cultures

Sophie

Losciuto, Gauthier

Dorban, Sébastien

Gabel, Audrey

Gustin, Claire

Hoenen,

Luc

Grandbarbe,

Paul

Heuschling,

Tony

Heurtaux

∗

LifeSciencesResearchUnit,FacultyofScience,TechnologyandCommunication,UniversityofLuxembourg,CampusLimpertsberg, 162A,AvenuedelaFaïencerie,L-1511,Luxembourg

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3January2012

Receivedinrevisedform19March2012 Accepted21March2012 Keywords: Brain Astrocytes Immunecells Inflammation Beta-amyloid

a

b

s

t

r

a

c

t

Microglia,theCNSresidentmacrophages,andastrocytes,themostabundantglialcellpopulation,are bothimplicatedinbrainpathologiesandcanexhibitapro-inflammatoryphenotype.Microglialcellsare knowntorapidlyandstronglyreacttobraininsults.Theywillpromoteastrocyteactivationandmay leadtoavicious,self-perpetuatingcycleofchronicinflammation.Toobtainabetterunderstandingof theindividualroleofbothcelltypes,primarycellsarefrequentlyusedininvitrostudies,butthepurity ofspecificcellculturesremainsrarelyinvestigated.Theaimofthisstudyistodeterminetheeffectof specificremovalofmicroglialcellsontheinflammatorypropertiesofdifferentglialcultures.Here,the removalofmicroglialcontaminationfrommixedglialculturestoobtainastrocyte-enrichedcultures wasachievedusingamagneticcellsortingapproach.Comparedtomixedcultures,weclearlyshowed thattheseenrichedculturesareonlyweaklyactivatedbypro-inflammatoryagents(lipopolysaccharide, interferon-!orbeta-amyloidpeptide).Thisfindingwasconfirmedusingtwice-sortedastrocyte-enriched culturesandmicroglia-freeculturescomposedofneurosphere-derivedastrocytes.Thus,wepresent evi-dencethatthemagnitudeofthepro-inflammatoryresponseislinkedtothepercentageofmicrogliain cultures.Duetotheirhighreactivitytovariousinsultsorpro-inflammatorystimuli,microglia-derived effectscouldbecreditedtoastrocytesinmixedglialcultures.Therefore,wehighlighttheimportance ofmonitoringthepresenceofmicrogliainglialculturessincetheycanaffecttheinterpretationofthe results,especiallywheninflammatoryprocessesarestudied.

1. Introduction

Inthecentralnervoussystem(CNS),glialcellshavesupportive

rolesforneuronsbutalsoactastheresidentimmunesystemcells,

engagingseveralinflammatoryprocessestodefendthebrainfrom

pathogensandhelpingittorecoverfromstressandinjury(Farfara

etal.,2008).However,inbrainpathologies,chronicglialactivation

isacommonevent,whichcouldpromoteaprogressive

dysfunc-tionandlossofneuronsintheCNS.Astrocytesandmicrogliaare

bothglialcellswhosephysiologicalrolesandresponsesupon

acti-vationarequitedistinct.Astrocytes,themostabundantglialcell

population,areessentialforbrainhomeostasisandneuronal

func-tion.Although astrocytes play a significantrole in maintaining

normalCNSfunctions,afterinjurytheseglialcellsmayexert

patho-physiologicalactionsleadingtoneuronaldamage(Sofroniewand

Vinters,2010).Microglialcells aretheresidentmacrophages in theCNS (Beutner etal., 2010; Hanisch and Kettenmann,2007; Dobrenis,1998). These cells represent the first line of defense

∗ Correspondingauthor.Tel.:+3524666446382;fax:+3524666446371. E-mailaddress:tony.heurtaux@uni.lu(T.Heurtaux).

againstpathogensorbraintissueinjury.Intheiractivatedstate,

theyhavebeneficialfunctionsessentialforneuronsurvival(Ekdahl

etal.,2009).Ontheotherhand,microglialoveractivationcangive

risetoprogressiveneurotoxicconsequences(LullandBlock,2010;

Blocketal.,2007).Microglialactivationcanalsopromoteastrocyte

activation,whichinturncanfurtheractivatemicroglia,thus

possi-blyleadingtoaviciouscycleofoveractivationthatmaycontribute

toachronicinflammatorystate,withcontinuouslyincreasing

pro-ductionofpro-inflammatorymediators(Liuetal.,2011;Vallejo

et al.,2010;Saijoetal.,2009;Henze etal.,2005;Zaheer etal., 2002).

Invitroexperimentsusingnear-pureglialculturesarenowadays

widelyusedinordertoavoidheterogeneouscell–cellinteractions

andtocharacterizecell-specificsignalingpathways.Thus,diverse

methodshavebeendevelopedforcellseparationandpurification

(forreview,seeDainiaketal.,2007).Actually,themostwidelyused

techniqueforharvestingasingleglialpopulationisdependenton

amechanicalshake-offmethod(FlodenandCombs,2007),butthis

iswroughtwithissuesasitoftenstillleavesamixtureof

differ-entglialcells.Newertechniquesarebasedonphysicalcriterialike

size,shape,anddensity.Theyincludefiltrationandcentrifugation

approaches(Fussetal.,2009;LaraandChalmers,2005;Graham,

(3)

2001),andalsoaffinityapproachesbaseduponbiochemicalcell

surfaceand biophysical characteristics(Choet al., 2010).Other

approachesaretheuseofanti-mitoticagentstoinhibitmicroglial

growthinprimaryastrocytecultures(Hambyetal.,2006a;Solenov

etal.,2004)orthedifferentialadherenceofvariousglialcellsoncell

cultureplates(Janaetal.,2007).Inthisstudy,weusedamorerecent

techniqueforprimarymicrogliaisolation(Mareketal.,2008).Based

onan antigen–antibody-mediatedmagnetic cellsorting system

usingCD11bMicroBeads,thistechniqueallowedtorapidlyobtain

pureastrocyteculturesaftermicrogliadepletion.

Our workfurtherstresses thegrowingconsensus that great

caremustbetakenwhenusingglialculturestoassessastrocyte

responsestoinflammatorystimuli,asevensmalllevelsofmicroglia

willhave a major influenceon theobserved effects. Therefore,

withoutarigorousqualitycheck,microglia-derivedeffectscould

beattributedtoastrocytes.Duetotheirhighpro-inflammatory

capacities,microgliarapidlydetectandstronglyrespondtoalarge

numberofstimuli.Thepresenceofmicrogliamustnotbe

underes-timatedandgreatcautionshouldbetakenwhenpreparingprimary

glialcultures(Saura,2007).Thus,weinvestigatedwhether

classi-calpro-inflammatorystimuli,suchasLPS,IFN!orbeta-Amyloid

(A") peptide,were able toactivate different types of glial cell

cultures:mixed glialcultures (calledMGC1),astrocyte-enriched

cultures(calledAEC-M1andAEC-M2),neurosphere-derived

astro-cytecultures,and primarymicroglia cultures.Weprovideclear

evidencethatthemagnitudeofthepro-inflammatoryresponsewas

dependentontheproportionofcontaminatingmicroglialcellsin

ourcultures.

2. Materialsandmethods

2.1. Mixedglialcultures

MixedglialcellcultureswerepreparedfromnewbornC57BL/6

mousebrains(Harlan,TheNetherlands)(Fig.1).Briefly,after

care-fullyremovingmeningesandlargebloodvessels,thebrainswere

pooledandthenmincedincoldphosphate-bufferedsaline(PBS)

solution.Thedissociationwascompletedbya10minincubation

in2mM-cold EDTA.Afterwashes and centrifugations,the cells

wereplatedandgrownincompletemedium(Dulbecco’sModified

EagleMedium(DMEM)supplementedwith10%fetalbovineserum

(FBS),100U/mlpenicillinand100#g/mlstreptomycin)at37◦_C_in

ahumidifiedatmospherecontaining5%CO2.Theculturemedium

waschangedafter3daysofculture.After8–10days,mixedglial

cul-tures(passage0orP0)hadreachedconfluence,andmicrogliawere

detachedbya30minshakingonarotaryshakerat200rpmat37◦_C.

Detachedcells, mainlymicroglia(Iba1-positivecells>95%)were

thenplatedinthesamemedium(astrocyte-conditionedmedium)

inahumidifiedatmospherecontaining5%CO2andwere

ready-to-use24hlater.Oligodendrocytesweresubsequentlydetachedfrom

themixedglialcultures(P0)by2daysofwashingandshakingsteps

onarotaryshakerat200rpmat37◦_C_in_a_humidified_atmosphere

containing5%CO2.Afterwards,theculturemediumwasremoved

andmixedglialcultureswerewashedwithPBSandincubatedwith

0.05%trypsin/0.53mMEDTAat37◦_C_for₅_min._Then,_the_trypsin

wasinactivatedbytheadditionofcompletemediumandthe

cellu-larsuspensionwascentrifugedat300×gat4◦_C_for₁₀_min._The_cell

pelletwasresuspendedincompletemediumandthenplated.After

6–7days,mixedglialcultures(P1),calledMGC1,hadreached

con-fluence.Todetachresidualmicrogliaandoligodendrocytes,these

cultureswereshakenagainfor2dayswith3washingsperday

onarotaryshakerat200rpmat37◦_C_in_a_humidified_atmosphere

containing5%CO2.Finally,freshcompletemediumwasaddedand

theseMGC1wereready-to-use.Cellcultureproductswere

pur-chasedfromInvitrogen(Scotland).

2.2. Magneticcellsortingforastrocyte-enrichedcultures

AsindicatedinFig.1,mixedglialcultures(P1)orMGC1,obtained

after the trypsinization step (described above), were further

worked-upbymagneticcellsorting(MACS).Aftertrypsinization

andcentrifugationat300×gat4◦_C_for₁₀_min,_the_cell_pellet_was

resuspendedina separationbuffercontainingPBS,pH7.2, 0.5%

bovineserumalbumin(BSA)and2mMEDTA.Thecellsuspension

wascentrifuged at 300×g at4◦_C _for ₁₀_min._Following

manu-facturer’sinstruction,1×107 _cells_were_resuspended_in₉₀_#l_of

bufferand10#lofCD11bMicroBeads(MicroBeadsconjugatedto

monoclonalratanti-mouseCD11bantibody,MiltenyiBiotec,The

Netherlands).Thesuspensionwasincubatedat4◦_C_for₂₀_min_with

gentlemixingafter10min.Duringtheincubationstep,the

CD11b-positive cells, microglia,weremagnetically labeledwithCD11b

MicroBeads.Then,cellswerewashedwiththeseparationbuffer

and centrifugedat300× gat4◦_C _for₁₀_min._The_cellular_pellet

wasresuspendedin500#lofseparationbufferfor1× 108cells.

ALScolumn(MiltenyiBiotec)wasfixedintotheMidiMACS

mag-neticseparationunit(MiltenyiBiotec)andwashed with3ml of

separationbuffer.Thecellsuspensionwasappliedonthecolumn

andthreewashingsof3mlofseparationbufferwereperformed

withthesamecollectingtube.Theunlabeled-cellfraction,mainly

astrocytes,waspassedthroughthecolumn.TheLScolumnwas

thendetachedfromthemagneticseparatorandflushedwith5ml

ofseparationbuffer.Thisstepwasperformedtocollect

CD11b-positivecells.Bothcellularfractionswerecentrifugedat300×gat

4◦_C_for₁₀_min._The_{astrocyte-enriched}_fraction_(called_AEC-M1),

correspondingtotheCD11b-negativefraction,wasresuspended

incompletemediumbeforeplating.After6–7days,AEC-M1were

confluentandready-to-use.TheCD11b-positivefraction,microglia,

wereplatedinastrocyte-conditionedmediumandwere

ready-to-use24hlater.

In ordertovastlyreducemicroglialcontamination,a second

magneticcellsorting(aspreviouslydescribed)waspracticedfrom

AEC-M1toobtainevenpurerastrocytecultures(AEC-M2).After

6–7days,AEC-M2wereconfluentandready-to-use.

2.3. Neurosphere-derivedastrocytecultures

Primaryculturesofneurosphereswereobtainedfrom

embry-onic murine neural stem cells derived from the subventricular

zone (SVZ)ofembryonicday14 (E14)C57BL/6 mouseembryos

as described previously (Grandbarbe et al., 2003). The SVZ of

embryonic brainwas extracted, triturated mechanically witha

fired-narrowedPasteurpipette.Dissociatedcellswerecentrifuged

at300×gfor5min,washedonceand thenplatedinnoncoated

6-wellplatesuntilneurospheresappeared.Neurosphereswere

cul-turedinNeurobasal-Amediumsupplementedwith1%B27without

vitaminA,2mMl-glutamine,100U/mlpenicillin,100#g/ml

strep-tomycin and 20ng/ml recombinant mouse Epidermal Growth

Factor(rmEGF).Theseneurosphereswerepassagedbychemical,

non-enzymaticdissociationevery5–6daysandreseededassingle

cellsatadensityof1×106_cells/ml._Neurospheres_were

differen-tiatedintoastrocytesbyplatingthemonpoly-l-ornithinecoated

flasksin DMEMcontaining10%FBS and 1%antibiotics(Crocker

et al.,2008).Theculture mediumwasrenewed after3 days of

differentiationand subsequentlyonce aweek.After6weeks of

differentiation,thecellswereconsideredtobecompletely

differ-entiatedintomatureastrocytes.

2.4. Celltreatments

Alltreatmentswereappliedonconfluentcultures.

Lipopolysac-charide (LPS 055:B5from Escherichiacoli, Sigma,Belgium) was

(4)

Brain tissue (newborn mice)

Preparation of cell suspension by trituration and EDTA incubation. Cells were allowed to adhere in complete medium On 3rd_{day, mixed glial}_cultur_{es (P0)}_{were wash}_ed After 8-10 days, mixed glial cultures (P0) were confluent.

Cells were shaken at 200 rpm at 37°C for 30 min

Cells in suspension (microglia) were

plated in astrocyte-conditioned medium Adherent cells were maintainedCultures were shaken again at in co 200mplete rpm at mediu 37°Cm. for 2 days with 3 washings per day

Trypsinizationstep Trypsinization step (Trypsin-EDTA)

Mixed glial cultures (P1) (called MGC1) were allowed to adhere incomplete medium

Magnetic cell sorting (CD11b MicroBeads antibody)

Astrocyte-enriched cultures (called AEC-M1)

were allowed to adhere in complete medium astrocMicroglia yte-conditiwere culturoned medied inum

CD11b-negative

fraction CD11b-positivefraction

p y

After 7 days, trypsinization and magnetic cell sorting using the

CD11b MicroBeads antibody

CD11b-negative fraction

Astrocyte-enriched cultures (called AEC-M2) were allowed to adhere in complete medium

g f

Fig.1.Schematicdrawingrepresentingthecellisolationprocess.

only1ng/mlonprimarymicroglia,sincea higherconcentration

is deleterious for these cells. IFN! (Hycult Biotechnology, The

Netherlands)wasusedat50U/ml.TheA"(1–42)peptide(Bachem,

Germany)wasinitiallydissolvedto5mMinDMSO,separatedinto

aliquotsinsteriletubesandstoredat−20◦_C._The_stock_aliquot_was

dilutedto0.5mMinDMEMcontaining1%antibiotics(100U/ml

penicillinand100#g/mlstreptomycin).ThisA"preparationwas

addeddirectlytoculturesatafinalconcentrationof5#M.

2.5. Cellularviabilityassay

Mitochondrialfunctionwasassessedasanindexofcellular

via-bilityusingtheMTT method.Thisassaymeasurestheabilityof

mitochondrialdehydrogenasestoconvert

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide(MTT)tocoloredinsoluble

formazan.Cellswereseededon48-wellplateataninitialdensity

of2×105_cells/well._After_treatment,_cells_were_incubated_for₃_h

at37◦_C_with_MTT_(0.35_mg/ml)_diluted_in_PBS._Then,_the_medium

wasremoved, andcells were lysedbyaddition of100% DMSO.

Absorbancewasmeasuredat540nmusinga microplatereader

(TECAN,Austria).Viabilitywasestimatedfromtheabsorbanceof

treatedversusuntreatedcells.

2.6. Flowcytometry

The MACS-basedsorting of microglialcells frommixed glial

cultureswasassessedbyflowcytometry.Cellswereharvestedby

trypsinization,centrifugedandresuspendedin3%BSAinPBS.For

intracellularmarkers,cellswerepreviouslyfixedand

permeabi-lizedinPBS-paraformaldehyde4%and0.01%ofTritonX100at4◦_C

for30min.Afterawashingstep,cellswereincubatedfor45min

withprimary antibodies:rabbit anti-Iba1(1:100,Biocare

Medi-cal,USA)orCy3-labeledmouseanti-GFAP(1:800,Sigma,Belgium).

Then,cellswerestainedwithCy2-conjugatedanti-rabbit(1:1000,

JacksonImmunoResearch,England).ThecellswerewashedinPBS

(5)

procedurewasrepeatedin parallelwiththecorresponding

iso-typecontrolantibodies(JacksonImmunoResearch).Thesamples

wereanalyzedonaflowcytometer(FACSCanto,BectonDickinson)

anddataanalysiswasperformedusingWinMDIsoftware(Scripps

ResearchInstitute,USA).

2.7. Immunofluorescence

Cells werecultured onpoly-l-ornithinecoatedcoverslips for

oneweekandthenfixedwithparaformaldehyde(4%inPBS)for

20minatroomtemperature,andpermeabilizedfor5mininPBS

containing0.3% Triton X100. Following3 washings in PBS,the

blockingstepwasrealizedwith3%BSAinPBSatroom

temper-aturefor30min.Thecellswerethenincubatedovernightat4◦_C

withCy3-labeledmouseanti-GFAP(1:800,Sigma)andrabbit

anti-Iba1(1:300,BiocareMedical).AfterwashingstepswithPBS,cells

wereincubatedwithCy2-conjugatedanti-rabbit(1:1000,Jackson

ImmunoResearch)at roomtemperature for onehour. The cells

werewashed3timeswithPBS,andstainedwithDAPI(1:5000)

for5min. Cellswerethen washed, mountedwithFluoromount

G(SouthernBiotech,USA),andobservedunderaLSM510META

invertedconfocalmicroscope(CarlZeissMicroImaging,Göttingen,

Germany)ata40-foldmagnification.

2.8. Real-timereversetranscription-polymerasechainreaction

(RT-PCR)analysis

TotalRNAwasisolatedfromglialculturesusingtheInvisorbTM

SpinCellRNAMiniKit(Invitek,Germany)accordingtothe

man-ufacturer’s protocol.Total RNAwas recoveredin elution buffer

and quantifiedfromthe absorbanceat 260nm.The puritywas

verifiedbyspectrophotometry(absorbanceratioat260nmversus

280nm).TheRNAelectrophoresisExperionsystem(Bio-Rad,CA)

wasusedtoconfirmtheabsenceofRNAdegradationandgenomic

contamination.ComplementaryDNA(cDNA)wassynthesizedfrom

RNAsamplesusing0.5#goftotalRNAandtheImProm-IIReverse

Transcription System with 0.5#g/reaction oligo(dT) as primer.

TheRNAand oligo(dT)weredenaturated at70◦_C _for₅_min_and

immediatelychilledonicefor at least5min.The reverse

tran-scriptionreactionwasperformedin a totalvolumeof 40#l by

mixing 2#l ImProm-IITM _reverse _{transcriptase,} ₂₅_mM _MgCl

2,

0.5mMofeachdNTP,and1#lofRecombinantRNasin®

Ribonu-cleaseinhibitorin ImProm-II5XReactionBuffer.Sampleswere

incubated for 1h at 42◦_C _and ₁₅_min _at ₇₀◦_C _to _inactivate

thereverse transcriptase. A 1#l aliquot of cDNA was used for

each amplification by real-time PCR and added to the

incuba-tionmixturecontaining 8#l of2XiQTM _SYBR _Green _Supermix,

0.8#l of each 12.5#M optimized forward and reverse primers

in a final volume of 20#l. Primer sequences (Table 1) were

designedusingtheBeaconDesignerSoftware(Bio-Rad).PCR

anal-ysiswere performed ona Bio-Rad iCycler (iQ5 Real-Time PCR

DetectionSystem)usinga2-stageprogramprovidedbythe

man-ufacturer: 3min at 95◦_C _followed _by ₄₀ _cycles, _consisting _of

denaturationat95◦_C_for₁₀_s_and_{annealing/extension}_at_a

tem-perature between 52.5◦_C _and _60.5◦_C _for ₃₀_s. _The _change _of

reporterfluorescence from each reaction tube was monitored.

Analysisof geneexpressionwasperformedusingthe

compara-tivethresholdcycle(Ct)method.Thethresholdcycleofeachgene

wasdetermined asPCRcyclesat whichanincrease in reporter

fluorescence above a baseline signal was observed (Table 2).

The target gene was normalized to the endogenous reference

gene, Rpl27 (a housekeepinggene coding for a ribosomal

pro-tein). The mRNA expression fold change was calculated using

theexpression2−ddCt_,_where_ddCt₌_(C_t_,_target₋_C_t_,_Rpl27₎_treated_sample

−(Ct,target−Ct,Rpl27)controlsample.

Table1

Sequencesofthedifferentreal-timePCRprimers.

Targetgene (accessionnumber)

Sequence(5′_–3′₎ _Product_size

Nos2(NM 010927) f:AGCCCTCACCTACTTCCTG r:CAATCTCTGCCTATCCGTCTC 100bp Ptgs2(NM011198) f:GCCTGGTCTGATGATGTATGC r:GAGTATGAGTCTGCTGGTTTGG 124bp Tnf(NM013693) f:GGTTCTGTCCCTTTCACTCAC r:TGCCTCTTCTGCCAGTTCC 107bp Il1" (NM 008361) f:GCTTCAGGCAGGCAGTATC r:AGGATGGGCTCTTCTTCAAAG 133bp Il6(NM031168) f:ACCGCTATGAAGTTCCTCTC r:CTCTGTGAAGTCTCCTCTCC 117bp Ccl2(NM011333) f:ACTCACCTGCTGCTACTCATTC r:GCTTCTTTGGGACACCTGCTG 97bp Ccl3(NM 011337) f:TTTGAAACCAGCAGCCTTTG r:CAGGTCAGTGATGTATTCTTGG 114bp Gfap(NM010277) f:GGTTGAATCGCTGGAGGAG r:CTGTGAGGTCTGGCTTGG 131bp Itgam(NM 008401) f:TGGACGCTGATGGCAATACC r:GGCAAGGGACACACTGACAC 94bp Aif1(NM019467) f:TTCCCAAGACCCACCTAG r:TCCTCATACATCAGAATCATTC 142bp Rpl27(NM011289) f:ACATTGACGATGGCACCTC r:GCTTGGCGATCTTCTTCTTG 111bp

Allreagentsforreversetranscriptionandreal-timePCRwere obtainedfromPromega(CA)andBio-Rad,respectively.

2.9. Measurementofpro-inflammatorymediatorrelease

Cells were seeded on 6-well plates at an initial density of 105_cells/well._After_6–7_days,_cultures_were_confluent_and ready-to-use.Thereleaseofnitrite,astablemetaboliteofnitricoxide(NO), wasmeasuredafter24and 48hofLPS,IFN! andA"exposure. The concentration of nitrite was determined in culture super-natantsbytheGriessassay.TheproductionofprostaglandinsE2 (PGE2)and TNF$wasmeasuredafter24hof LPS,IFN!and A" exposure.PGE2 andTNF$releaseweremeasuredinthemedium of thedifferentcultures witha commercially availableEnzyme ImmunoAssaykit(AssayDesigns,USA),andasandwichEnzyme LinkedImmunoSorbentAssay(R&DSystems,USA),respectively. TheproteincontentwasdeterminedusingtheDyeReagent (Bio-Rad)bymeasuringtheabsorbanceat595nm,usingbovineserum albuminstandards.

2.10. Statisticalanalysis

Forcomparisonofmeansbetweentwodifferenttreatments, sta-tisticalanalysiswasdonebyStudent’st-test(two-sampleassuming equalvariances).Forcomparisonof multipletreatments,results were analyzed by anANOVA followed by a Fisher’s exact test. Differencesbetweengroupswereconsideredassignificantwhen p-valueswerelessthan0.05.Resultsareexpressedasmean±SEM fromatleastthreeindependentexperiments.

3. Results

Following the experimental procedure described in Fig. 1,

we have prepared mixed glial cultures (P0) from thebrains of

newborn mice. From these cultures, we were able to prepare

other glial cultures, such as mixed glial cultures (MGC1), but

alsoastrocyte-enrichedcultures(AEC-M1),twice-sorted

astrocyte-enrichedcultures(AEC-M2)andmicroglialcultures.Inthisstudy,

we have also generated microglia-free cultures composed of

(6)

Table2

Thresholdcycle(Ct)valueofthetargetgenesinthedifferentcultures.

Targetgene(accessionnumber) MGC1 AEC-M1 Neurosphere-derivedastrocytes Primarymicroglia

Nos2(NM010927) 36.0 36.0 36.0 37.0 Ptgs2(NM011198) 28.0 27.5 33.5 30.0 Tnf(NM013693) 29.0 36.0 34.5 26.5 Il1" (NM 008361) 30.5 35.5 36.5 29.0 Il6(NM 031168) 31.5 31.5 37.0 35.0 Ccl2(NM011333) 25.0 26.5 28.0 25.0 Ccl3(NM011337) 27.0 35.0 31.5 22.0 Gfap(NM010277) 21.0 21.0 20.5 – Itgam(NM008401) 29.2 34.4 36.0 22.6 Aif1(NM 019467) 24.6 30.8 38.2 21.8 Rpl27(NM 011289) 20.0 20.5 20.5 20.0

3.1. Analysisofthedifferentglialcellculturepreparations

Immunocytochemistryanalysis revealed that the number of microglialcells (Iba1-positive cells) in MGC1 wasmuchhigher comparedtotheonescontainedinAEC-M1(Fig.2).Mouse

astro-cytesobtainedbythisseparationprocessshowedtypicalastrocyte

morphologyaswellasastrocyticmarkerexpression(GFAP),which

wasslightlylowerthaninMGC1.Inbothcultures,somecellswere

negativeforbothIba1andGFAPmarkers.Furthermore,

quantita-tivedatahavebeenobtainedbyFACSanalysis(Fig.3).Asshownin

Fig.3A,aMACSseparationincreasedtheGFAP-positivecellsfrom

about60%inMGC1to70%inAEC-M1.Thepercentageofmicroglial

cells(Iba1-positivecells)hasalsobeendeterminedin both

cul-tures.Beforecellsortingseparation,Iba1-positivecellsrepresented

9–10%oftheculture(Fig.3B),whereasIba1-positivecellnumber

decreasedto1–1.5%ofthetotalcellsinenrichedcultures(Fig.3C).

CellsortingbyCD11bMicroBeadswasusedtoobtain

astrocyte-enrichedcultures,butalsomicroglialcultures(>95%Iba1-positive

cells,datanotshown).

3.2. Effectsofpro-inflammatorystimuli:assessmentofmRNA

levels

In order to evaluate the consequences of the presence of

microglialcellsinourastrocytecultures,pro-inflammatory

condi-tionswereinducedbytreatingMGC1andAEC-M1withLPS,IFN!

andA".Pro-inflammatorygeneexpressionswereassessedby

real-timePCR(Fig.4).

InMGC1,a6hLPSexposurestronglyinducedtheexpression

ofNos2(Fig.4A),Ptgs2(Fig.4B),Tnf(Fig.4C),Il1"(Fig.4D)and

Il6(Fig.4E)aswellasCcl2andCcl3chemokines(datanotshown).

InAEC-M1,wherethepercentageofmicroglialcellswasabout

10-foldlowerthaninMGC1,theexpressionofthesepro-inflammatory

geneswasmuchlowercomparedtomixedcultureresponses:Nos2

(−90%),Ptgs2 (−85%),Tnf(−95%),Il1"(−97%),Il6(−95%),Ccl2

(−50%,datanotshown)andCcl3(−90%,datanotshown).

IFN!andA"werelesspotentthanLPSinbothglialcultures,

buttheireffectswerestilldifferentbetweenMGC1andAEC-M1

cultures.InMGC1,IFN!andA"inducedNos2expression(Fig.4F)

(7)

Fig.3.Quantitativeassessmentofastrocyteandmicrogliapopulationsbyflowcytometry.Thedifferentglialcellpopulationswereimmunostainedforflowcytometry analysis.GFAP(A)andIba1-positivecells(BandC)weredeterminedinbothMGC1(greenevents)andAEC-M1(redevents).Fornegativecontrol(greyevents),thesame stainingprocedurewasrepeatedwiththesecondaryantibodiesalone.DataanalysiswasperformedusingWinMDIsoftware(ScrippsResearchInstitute,USA).Thisisa representationofoneoutofthreeexperiments.Allexperimentsshowedsimilarprofiles.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderis referredtothewebversionofthisarticle.)

byrespectively25-foldand5-foldcomparedtothecontrollevel.On

thecontrary,aftermagneticseparation,anIFN!exposureinduced

lowerNos2expression (4.5-fold),whereas A"had noeffect on

it.Inboth conditions,Ptgs2up-regulation(Fig.4G) wassimilar.

ControlexpressionlevelsofTnfandIl1"(respectivelyFig.4Hand

I) werehigher inMGC1 due toincreased basal expression

lev-elsinmicrogliacomparedtoastrocytes(Table2).Thus,inMGC1,

IFN!andA"up-regulatedTnfbyrespectively4.4-foldand2-fold

(Fig.4H).A"alsoincreasedIl1"expression(4-fold)contraryto

IFN!,whichdecreasedIl1"expression(−20%).InAEC-M1,these

two pro-inflammatory compounds had different effects onthe

expressionof TnfandIl1". An IFN!exposureup-regulated Tnf

expressionby1.6-foldwhereasA"hadnoeffect.However,anA"

exposureup-regulatedIl1"expression(2-foldinduction)whereas

IFN!hadnoeffect.IFN!andA"alsoinducedahigherIl6

overex-pressioninMGC1comparedtoAEC-M1(Fig.4J).

3.3. Comparisonwithneurosphere-derivedastrocytecultures

Neurosphere-derivedastrocytesareconsideredaspure

astro-cytes. In response to an inflammatory stimulus (Fig. 5),

neurosphere-derivedastrocytesreactedtoTNF$(datanotshown)

andIFN!exposure.ThepresenceofIFN!increasedgene

expres-sionlevels of Nos2, Ptgs2, Tnf and Il6 (respectivelyFig. 5A–D)

but,incontrasttoAEC-M1cultures,LPSexposuredidnotincrease

Nos2,Tnf,andIl6expression.Moreover,aLPStreatmentinduced

alittleincreaseofCOX2expression(2.2-fold).AfterIFN!andA"

treatments,theactivationprofileobservedinneurosphere-derived

astrocytes wasthesame than theone foundin AEC-M1.

How-ever,Il1"expressionwasnotdetectableinneurosphere-derived

astrocytes.Intheseconditions,theGfapexpressionlevelwasnot

modulatedbypro-inflammatoryactivations(Fig.5E).

3.4. Comparisonwithprimarymicrogliacultures

ComparedwithMGC1andAEC-M1,primarymousemicroglia

were highly sensitive to LPS (1ng/ml), IFN! (50U/ml) and A"

(5#M)exposures(Table3).Asexpected,primarymicrogliashowed

Table3

Pro-inflammatoryphenotypeinprimarymicroglia.

(8)

MGC1 AEC-M1

A

3000 ss ion

*

#

F

40 ss ion

*

# 0 1000 2000 Ctrl LPS Nos2 mRNA expre s fold change

*

0 10 20 30 Ctrl IFNγ Aβ Nos2 mRNA expre s fold change

*

#

G

1 2 3 R NA express ion ldc ha ng e

*

B

50 75 100 RNA express ion ld change

*

# 0 1 Ctrl IFNγ Aβ Ptgs2 mR fol 0 25 Ctrl LPS Ptgs2 mR fo

*

C

600

_*

#

H

4 5

*

# # 0 200 400 Ctrl LPS Tnf mRNA expression Tnf mRNA expression fold change

*

fold change Ctrl IFNγ Aβ 0 1 2 3 4

*

D

fold change 250 500 750 1000

*

#

I

2 3 4 5 ld change

_*

#

*

# Il1 β mRNA express ion Il1 β mRNA express ion f Ctrl LPS 0 250

*

E

ess ion ge 500

*

# 0 1 Ctrl IFNγ Aβ fo

*

J

*

6 8 ress ion ge # # Il6 mRNA expr e fold chang Ctrl LPS 0 250

*

0 2 4 Ctrl IFNγ Aβ Il6 mRNA exp r fold chang

*

_*

Fig.4. Evaluationofpro-inflammatorygeneexpressions.Pro-inflammatorygeneNos2(AandF),Ptgs2(BandG),Tnf(CandH),Il1" (DandI)andIl6(EandJ)expressions weredeterminedinMGC1andAEC-M1after6hofLPS(1#g/ml),IFN! (50U/ml)andA"(5#M)exposure.Thetranscriptionlevelswereanalyzedbyreal-timePCR.Control expressionlevelswerefixedat1.0inmixedculturecondition.AllgeneexpressionswerenormalizedtoRpl27expressionlevel.Resultsaregivenasmean±SEM(n=5 independentexperiments).*p<0.05,significantlydifferentfromcontrol;#_p_<_0.05,_{significantly}_different_between_MGC1_and_AEC-M1.

amuchstrongerinflammatoryresponsethananyoftheothercell populations.Thethreestimuliinducedhighexpressionlevelsof Nos2,Ptgs2,TnfaswellastheCcl2chemokine.However,anIFN! exposurereducedIl1" andCcl3expressionincontrasttoLPSand A"treatments.

3.5. Effectsofpro-inflammatorystimuli:assessmentof inflammatoryproteinrelease

(9)

A

15 20

*

0 5 10 LPS Ctrl IFNγ Aβ

Nos2 mRNA expres

sion fold change

B

1 2 3 fold change

*

_*

0 LPS Ctrl IFNγ Aβ Ptgs2 mRNA expres sion

C

2 3

*

0 1 LPS Ctrl IFNγ Aβ Tnf mRNA expression fold change

D

*

0 1 2 3 4 5

Il6 mRNA expression

fold change

E

1.0 1.5 Ctrl LPS IFNγ Aβ 0.0 0.5 LPS Ctrl IFNγ Aβ Gfap mRNA express ion fold change

Fig.5. Effectofpro-inflammatorycompoundsonneurosphere-derivedastrocytes geneexpression.Pro-inflammatorygeneNos2(A),Ptgs2(B),Tnf(C)andIl6(D) expressionsaswellasGfapexpression(E)weredeterminedinneurosphere-derived astrocytesafter6hofLPS(1#g/ml),IFN!(50U/ml)andA"(5#M)exposure.The transcriptionlevelswereanalyzedbyreal-timePCR.Controlexpressionlevelswere fixedat1.0.AllgeneexpressionswerenormalizedtoRpl27expressionlevel.Results aregivenasmean±SEM(n=3independentexperiments).*p<0.05,significantly differentfromcontrol.

(Fig.6).Theproductionofthesepro-inflammatorycompoundswas

higherunderLPSconditionscomparedtoIFN!orA"exposures.

LPS,IFN!andA"exposuresinducedanincreaseofnitritelevels

inMGC1at24hand48h(Fig.6A).InAEC-M1,nitritereleaselevels

werelowerthaninMGC1.ALPStreatmentslightlyincreasedthe

productionofnitritesafter24h(2-fold),butnotafter48h.IFN!or

A"exposuresdidnotpromotenitritesproductionatboth24and

48hinAEC-M1.

AnincreaseofPGE2(Fig.6BandC)andTNF$release(Fig.6Dand

E)wasobservedinMGC1after24hofLPS,IFN!andA"exposure.In

AEC-M1,thereleaseofprostaglandinsandTNF$inducedbyaLPS

treatmentwasstronglylower(respectively−95%and−99%)than

inMGC1.AnIFN!orA"exposurewasunabletopromotePGE2and

TNF$releasesinAEC-M1.

Inaddition,secretedamountsofnitrite,PGE2 andTNF$were

similarwhencellsweretreatedfor24hwithIFN!orA"inMGC1

orAEC-M1.

3.6. Timedependenteffectsofamicroglialcontamination

AEC-M1werestudieduntil3weeksafterplating (Fig.7).As

showninFig.7A,thenumberofmicroglialcellsincreasedfrom

7to21daysofculture.Aspreviouslydescribed,theGFAPstaining

waslowat7daysbuthigherafter21dayswhenmicroglialcells

(Iba1staining)werealsonumerous.A6hLPSexposure(Fig.7B)

increasedNos2,Ptgs2,TnfandIl1" expressioninatime

depend-entmanner.Thereby,thepro-inflammatorystatewashigherafter

21days,whichcoincidedwithanelevatednumberofIba1-positive

cells.

3.7. Comparisonbetweensingle-anddouble-sortedglialcultures

MGC1werealsocomparedtotwice-sortedglialcultures,called

AEC-M2(Fig.8).ItgamandAif1aregenescodingrespectivelyforthe

twoproteinsCD11bandIba-1,exclusivelyexpressedinmicroglia

butnotinastrocytes.AEC-M2presentednoIba1-immunostaining

(Fig.8A)aswellasnodetectablemicroglialmarkers(Itgamand

Aif1) mRNA expression (Fig. 8B). Compared to MGC1, AEC-M2

expressedlowerGFAPexpressionlevels(Fig.8AandB).Asaresult

oftheabsenceofmicroglialcells,apro-inflammatoryLPSexposure

hadonlyminoreffectsonAEC-M2andwassubsequentlynotable

toinducearealpro-inflammatorystate(Fig.8C).

4. Discussion

Morethan35yearsago,BooherandSensenbrennerdevelopedin

vitromethodsforbraincellscultures(BooherandSensenbrenner,

1972).Fordecades,thesemethodshavebeenusedbytheresearch

community.However,inthelastfewyears,scientistshavenoticed

that, due to theirhigh reactivity potential, microglialcell

con-taminationcouldaltertheexperimentalresponseobtainedfrom

primaryastrocyticcultures.Forthepurificationofglialcells,several

protocolsarefrequentlyfoundintheliterature,likecoating,

sub-culturingorshakingmethods.Toreducethepresenceofmicroglia

orothercontaminatingcells,cytosinearabinoside,aspecifictoxin,

anti-mitoticorlysosomotropicagentscanalsobeemployed(Saura,

2007;Hambyetal.,2006a).Themechanicalshake-offmethodisthe

mostwidelyused,whichallowsrecoveringmicroglialcellsthatare

presentabovetheastroglialmonolayer.However,withthis

proce-dure,theexclusionofmicroglialcellsfrommixedglialcellcultures

isincomplete.Indeed,thisshakingmethodappearstobeinefficient

toremovemicroglialcellslocatedbeloworinsidetheastroglial

monolayer.

Here, we have compared mouse MGC1 (twice shaken and

washed glial cultures), AEC-M1(magnetic cell sorted cultures),

AEC-M2 (twice-sorted cultures), neurosphere-derived astrocyte

culturesandprimarymicrogliacultures.Fromthemixedglial

cul-tures, magnetic cellseparationallows a single-step isolation of

microglialcells(CD11b-positivefraction),butalsoAEC-M1

(CD11b-negativefraction).In thisstudy,thesedifferenttypesofculture

methodsallowedustoevaluatetheimportanceofthepresenceof

microglialcellsintheestablishmentandanalysisofan

(10)

A

800 _24h 48h

*

Nitrite lev els (% of control) 200 400 600

*

#

*

* *

*

Ctrl LPS IFNγ Aβ Ctrl LPS IFNγ Aβ

0 1 M -C E A 1 C G M

B

# 8000 12000 entration rotein )

*

C

300 400 500 entration rotein )

*

# 0 4000 PGE 2 conce (pg/mg pr

*

Ctrl LPS Ctrl LPS 0 100 200 PGE 2 conc e (pg/mg pr

Ctrl IFNγ Aβ Ctrl IFNγ Aβ

1 M -C E A 1 C G M 1 M -C E A 1 C G M

D

10000

*

#

E

400

*

Fα α concentration (pg/mg protein) 50 6000 8000 100 200 300 N Fα concentration (pg/mg protein)

*

# TN 0 25

*

Ctrl LPS Ctrl LPS MGC1 AEC-M1 0 TN #

Ctrl IFNγ Aβ Ctrl IFNγ Aβ

MGC1 AEC-M1

Fig.6. Measurementofpro-inflammatorymediatorreleases.Nitrite(A),PGE2(BandC)andTNF$(DandE)releaseswerequantifiedafter24hand48h(fornitritesonly)

ofLPS(1#g/ml),IFN!(50U/ml)orA"(5#M)exposureinsupernatantofMGC1andAEC-M1.Resultsaregivenasmean±SEM(n=3independentexperiments).*p<0.05, significantlydifferentfromcontrol;#_p_<_0.05,_{significantly}_different_between_MGC1_and_AEC-M1.

Thefirstpartofthisworkaimedatdeterminingthe

propor-tionofmicrogliainMGC1andAEC-M1.Byimmunocytochemistry,

weconfirmedthatIba1-positivecellsarepresentinmouseMGC1

evenaftertwoshakingandwashingsteps.Conversely,afterCD11b

magneticseparation,theproportionofIba1-positivecells inthe

negativefraction,correspondingtotheAEC-M1,was,asexpected,

muchlowerthaninmixedcultures.Theimmunostainingofthe

glialfibrillaryacidicprotein(GFAP),anintermediatefilament

com-ponentofastroglialcells(Bramantietal.,2010;Röhletal.,2007),

wasslightlyhigher inMGC1 thanin AEC-M1.GFAPis a tightly

regulatedprotein,whoseexpressionisinducedbymultiple

fac-torssuchasbraininjuryanddiseases(MiddeldorpandHol,2011).

Removingmicroglialcellsfromculturepromotedadecreaseofthe

basal levelsofsecreted compoundslikeTNF$. Thus,a decrease

oftheastrocyticreactivityrelatedtoadown-regulationofGFAP

expressionwassubsequentlyobserved.Theamountofmicroglial

cellsinglialcultureswasaccuratelydeterminedbyflow

cytom-etry experiments. A CD11b magnetic separation decreasedthe

Iba1-positivecellsfrom9to10%inMGC1to1–1.5%inAEC-M1.

Con-sequently,theGFAP-positivecellsincreasedfrom60%inMGC1to

70%inAEC-M1.Theseresultsareinaccordancewithrecent

assess-mentsofglialpopulationsincultures(Mareketal.,2008).Besides,

cells thatwerenegativefor microglialand astroglialmarkersin

(11)

Fig.7. Time-dependenteffectofamicroglialcontamination.AEC-M1wereculturedfor7,14and21daysafterCD11b-magneticcellsorting.Thesecellswerelabeled withanti-GFAPantibody(red)incombinationwiththemicroglialmarkerIba1(green)andcounterstainedwithnuclearstainDAPI(blue)atthedifferenttimepoints(A). Pro-inflammatorygeneNos2,Ptgs2,TnfandIl1"expressions(B)werealsodeterminedintheseculturesafter6hofLPS(1#g/ml)exposure.Thetranscriptionlevelswere analyzedbyreal-timePCR.Controlexpressionlevelswerefixedat1.0.AllgeneexpressionswerenormalizedtoRpl27expressionlevel.Resultsaregivenasmean±SEM (n=3independentexperiments).*p<0.05,significantlydifferentfromcontrol.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothe webversionofthisarticle.)

oligodendrocytes,butalsoGFAP-negativeastrocytes.Infact,ithas

beenreportedthatsomeastrocytespopulationsinhealthyCNSdo

notexpress,orexpressatlowlevel,GFAP(SofroniewandVinters,

2010;Sofroniew,2009;Buffoetal.,2008).

Microglia, the most reactive cells against pro-inflammatory

stimuli,detectandrespondrapidlytopro-inflammatorytriggers

(Lull and Block, 2010; Kim and Joh, 2006). Based on previous

papers,astrocyte-mediatedeffectsarecompletelymaskedwhen

culturescontainalargepopulationofmicroglia.Forthisreason,

we agree with theauthors who have underlined the necessity

to minimizethe proportion of microglial cells in glial cultures

(Mareketal.,2008;Saura,2007;Hambyetal.,2006a,b;Hewett

etal.,1999).Thus,inthiswork,wehavemadeadirectcomparison

betweencultureswithaknownpercentageofmicroglia

contam-inationandcultureswithminimalornomicroglia.Toinducean

inflammatoryenvironment,wetreatedourglialcultureswith

pro-inflammatorycompounds,knowntopromotecellularactivation

(12)

Fig.8. ComparisonofMGC1withAEC-M2.Bothglialcellscultureswerelabeledwithanti-GFAPantibody(red)incombinationwiththemicroglialmarkerIba1(green)and counterstainedwithnuclearstainDAPI(blue)(A).TranscriptionlevelsandCtvaluesoftheastrocyticmarker(Gfap)aswellasmicroglialmarkers(ItgamandAif1)were

evaluatedbyreal-timePCRincontrolconditioninbothglialcultures(B).CultureswerealsotreatedwithLPS(1#g/ml)for6h.Thetranscriptionlevelsofthepro-inflammatory genesNos2,Ptgs2,TnfandIl1"wereanalyzedbyreal-timePCR(C).Controlexpressionlevelswerefixedat1.0.AllgeneexpressionswerenormalizedtoRpl27expression level.Resultsaregivenasmean±SEM(n=3independentexperiments).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtotheweb versionofthisarticle.)

factorkappaB(NF%B)pathway-inducer(LPS)(CarmodyandChen,

2007),ajanuskinase-signaltransducerandactivatorof

transcrip-tion(JAK-STAT)pathway-inducer(IFN!)(Goughet al.,2008)as

wellas thebeta-amyloid peptide(1–42) (A") in anoligomeric

conformation(Heurtauxetal.,2010).Inrecentstudies,wehave

demonstratedthatLPS,IFN!,aswellastheoligomericformofthe

A"peptidewereabletostronglyactivatemicroglialcells(Heurtaux

etal.,2010;Micheluccietal.,2009).Atthetranscriptionallevel,

we haveobserved that 6hofLPSexposurein MGC1induced a

higherpro-inflammatorystatethananIFN!orA"exposure.This

pro-inflammatorystatusappearedtobelowerinAEC-M1where

microglialcells were almostabsent.In theseAEC-M1,a 6hA"

exposurewasunabletoup-regulateNos2,Ptgs2and TnfmRNA

expressionandonlyslightlyinducedIl1"andIl6expression.Thus,

ashortsingleA"treatmenthadpooreffectsonAEC-M1

(13)

havealsoobservedthatpro-inflammatorymediators(nitrites,PGE2

andTNF$)releaseswerestronglydecreasedinAEC-M1compared

toMGC1bothtreatedwitheitherLPS,IFN!orA".Interestingly,

resultsobtainedwithA"clearlyshowedthata shorttreatment

was unable to activate AEC-M1. This is in accordance with a

recentstudy(Ebenezeretal.,2010).Inthiswork,wehaveused

anoligomericA"preparation,which isknowntostrongly

acti-vate microglialcells. These results supportthe hypothesis that

activationof astrocytes aroundA" depositsin Alzheimer’s

dis-easemaybeduetopriormicroglialactivationinducingfinallya

chronicinflammatorystate.Itiswelldocumentedthatactivated

microgliarepresentamajorsourceofinflammatoryfactors.Inthis

study,wehavedescribedthatprimarymicrogliastronglyreacted

toLPSexposure(Table3).Thepresence of IFN!or A"peptide

alsoinducedapro-inflammatoryphenotypeinprimarymicroglial

cells.Microglia appearedtobemore sensitivetoLPS,IFN! and

A" treatmentsthanAEC-M1andMGC1.Thestrongup-regulation

ofthe pro-inflammatory genes underlinedthe importance of a

microglial contamination even if the percentage of these cells

islowerthan 10%of thetotalcells in culture.Thus,toconfirm

theimportanceofmicrogliaintheinitiationofaninflammatory

environment,we have alsorealizedacontrol byusing cultures

devoidofmicroglialcells.Thesemicroglia-freeastrocytecultures

wereobtainedafterdifferentiationofneurospheres(Crockeretal.,

2008). Challenging these cultures with either LPS, IFN! or A"

had onlylittle effect ontheup-regulation of pro-inflammatory

genes.With the exception of LPStreatment, which is a strong

microglialactivator,theinflammatoryresponse in AEC-M1and

neurosphere-derivedpureastrocytecultures areobviously very

closed.Thus,both culturesrepresentsimilarinvitromodelsfor

astrocytestudies.Theseresultsareinaccordancewithpublished

work(Crockeretal.,2008).Inthesameway,Hambyetal.(2006a)

reportedamethodforeradicatingmicrogliafromprimaryglial

cul-tures.Theauthorstreatedconfluentastrocytemonolayerswitha

mitoticinhibitor(cytosine"-d-arabinofuranoside)followedbya

briefexposuretohighconcentrationsofalysosomotrophicagent

(l-leucinemethyl ester, LME). By this method, they generated

highlypurifiedastrocyteswithouttheneedforcellpassage.Their

resultsarequitesimilartoourresultsandconfirmthenecessityto

eliminatemicrogliafromglialcultures.However,thismethodusing

theLMElysosomotrophicagentonlypermitstoobtainnear-pure

astrocytecultureswhenourmagneticseparationpermitstoobtain

rapidly both near-pure astrocyte cultures and pure microglial

cultures.

Bacterial endotoxin receptors, mainly Toll-like receptor 4

(TLR4),areimplicatedinLPSbioactivity(Perietal.,2010).However,

intheseneurosphere-derivedastrocytes,theLPS-TLR4signaling

pathwaydoesnotseemtobeoperational.Thisobservationcould

belinkedtothelowlevelofTLR4expressioninastrocytes(Mishra

etal., 2006; Jacket al., 2005).TLR4 receptor is documentedto

bepresentoncirculatingmonocytes,macrophagesandother

sys-temicimmune cells.Wehavealsodemonstrated thatmicroglia

stronglyexpressTLR4whereasastrocytesdonotexpressit(data

notshown).Thesedataareinaccordancewithapreviouswork

(Lehnardtetal.,2002).Moreover,duetothehighTLR4expression

levelinmicroglia(Buchananetal.,2010;Mishraetal.,2006;Jack

etal.,2005),treatingglialcultureswithLPScouldrepresentaway

toconfirmtheirpresence.Furthermore,thebasalexpressionlevels

ofpro-inflammatorygenes(Tnf,Il1"andCcl3),aswellasthoseof

specificmicroglialmarkers(ItgamandAif1)arehigherinprimary

microgliathaninMGC1(Table2).InAEC-M1andin

neurosphere-derivedastrocytes,theexpressionlevelsareevenlower(threshold

cycleCt>30).Accordingtothesedata,thelevelofmicroglialcell

contaminationcouldalsobeevaluatedbyasimplecomparisonof

thethresholdcycleofthesepro-inflammatorygenesormicroglial

markersinbasalconditionsincultures.

Wehavealsodescribedtime-dependenteffectsofamicroglial

contamination.AEC-M1werestudiedupuntil3weeksafterplating.

ThepercentageofIba1-positivecellsincreasedinatime

depend-entmannerinthesenon-treatedconfluentglialcultures.Possibly

asaresultoftheincreasednumberofmicroglia,theGFAP

expres-sion level was also enhanced. This is in accordance with our

observations, wherewe described thatastrocytic reactivityand

GFAPexpressionwereincreasedinthepresenceofmicroglialcells

(Fig.2).Moreover,aLPSexposureinducedatimedependent

pro-inflammatorystate,which washigherafter3 weeksof culture,

andcoincidedwiththepresenceofahighnumberof

contaminat-ingmicroglialcells.Finally,wehavecomparedMGC1andAEC-M2

(twice-sortedglialcultures).TheAEC-M2presentedalowerGFAP

expressionlevelandnodetectablemicroglialmarkersexpression.

Furthermore,aLPSexposurewasnotabletoinduceareal

pro-inflammatorystate,thusconfirmingtheabsenceofmicroglialcells

withthisculturingprocedure.

In our conditions, we clearly show that microglia-depleted

cultures (AEC-M1/M2 and neurosphere-derived astrocytes) are

weakly activated by pro-inflammatory agents. The presence of

microgliaappearstobecrucialintheestablishmentofan

inflam-matory state. There is growing interest about the role of glial

cellsinneurodegenerativepathologies(Amoretal.,2010;Hamby

and Sofroniew,2010; Perryetal.,2010).On onehand,the

iso-lation of glial cells allows studying theresponsiveness of each

celltypetopro-inflammatoryagents.Ontheotherhand,theuse

of mixed cultures couldreflect the physiologicalconditionand

couldbeusefultoobserveglobalmodificationsandcellular

inter-actionscharacterizingCNSdisorders.Microglialcellsrapidlyand

stronglyreacttobraininsults.Duetotheirhighreactivitytovarious

aggressionsorpro-inflammatorystimuli,microglia-derivedeffects

mightbecreditedtoastrocytesinmixedcultures.Inthese

con-ditions,thesubsequentreactiveastrocytesinteractinacomplex

mannerwithmicrogliaandcanexertpro-inflammatorybutalso

anti-inflammatoryeffects(Fulleretal.,2010;Zhangetal.,2010).

In conclusion,ourresultssuggest thenecessitytorigorously

checkthepurityofglialcultures.Attentionshouldbepaidtothe

presenceofmicrogliainglialculturesbecauseoftheirpotential

alterationoftheresults,especiallywheninflammatoryprocesses

areinvestigated.Forthatreason,themagneticcellsorting

technol-ogyappearstobeanefficientandrapidmethodtolimitmicroglial

contaminationin glial cultures. Inaddition, itallows torapidly

obtainnear-pureastrocyteculturesbutalsopuremicroglial

cul-tures.

Conflictofintereststatement

Noconflictsofinterestexist.

Acknowledgements

This work was financed through grants from the National

Research Fund of Luxembourg and the University of

Luxem-bourg.WethankDrs.A.MichelucciandM.Buttiniforeditingthe

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