The Mouse Colony Forming Cell CFC Assay using Methylcellulosebased Media.docx

The Mouse Colony Forming Cell CFC Assay using Methylcellulosebased Media.docx

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TheMouseColonyFormingCellCFCAssayusingMethylcellulosebasedMedia

TheMouseColonyFormingCell（CFC）AssayusingMethylcellulose-basedMedia

Thecolonyformingcell（CFC）assay,alsoreferredtoasthemethylcelluloseassay,isaninvitroassayusedinthestudyofhematopoieticstemcells.Theassayisbasedontheabilityofhematopoieticprogenitorstoproliferateanddifferentiateintocoloniesinasemi-solidmediainresponsetocytokinestimulation.Thecoloniesformedcanbeenumeratedandcharacterizedaccordingtotheiruniquemorphology.

Pleasereadtheprotocolinitsentiretybeforestarting.

SuppliesRequired

Reagents

∙Cellsderivedfrommousebonemarrow,spleen,peripheralblood,orfetalliver.Miceareroutinelyusedbetween6-12weeks.

∙Iscove’sModifiedDulbecco’sMedia（IMDM）

∙FetalBovineSerum

∙IMDM/2%FetalBovineSerum

∙（Optional）FlowCytometryMouseLyseBuffer（R&DSystems,Catalog#FC003orequivalent）

Materials

∙100mmculturedishes

∙35mmculturedishes

∙15mLcentrifugetubes

∙10mLsyringes

∙3mLsyringes

∙5mLvials

∙16gauge,1½inchneedle

∙14gaugelaboratorypipettingneedle（Popper&Sons,Catalog#7941orThermoFisherScientific,Catalog#14-825-16M）

∙Serologicalpipettes

∙Pipettesandpipettetips

Equipment

∙37°Cand5%CO2humidifiedincubator

∙Centrifuge

∙Vortexmixer

∙Hemocytometer

∙InvertedMicroscope

Reagent&MediaPreparation

Note:

Steriletechniqueisrequiredwhenhandlingthereagents.

∙Methylcellulose-basedMedia-Thawthebottleofmediaat2-8°Covernight.Afterthemediaiscompletelythawed,shakethebottlevigorouslytothoroughlymixthecontents.Allowairbubblestoescapebyplacingthebottleeitheratroomtemperatureorat2-8°Cfor0.5-1hour.

Aliquottheexactamountofmediarequiredforasingleexperiment（Table1）intosterile5mLvialsusingasterile14gaugelaboratorypipettingneedleanda10mLsyringe.

Note:

Duetothehighviscosityofmethylcellulosemedia,theuseofasyringeisnecessarytoaccuratelymeasurevolume.The14gaugelaboratorypipettingneedlereferredtointheSuppliesRequiredsectionisrecommendedduetoitslargerdiameter.Theneedleisautoclavableandreusable.

Storethealiquotsat-20°Cinamanualdefrostfreezeruntiluse.Donotusepastthekitexpirationdate.

Table1.DuetothedifferentrequirementsforeachproductintheCFCassay,therecommendedvolumeforeachislisted.

Reagent

Catalog#

Volumex2*

Volumex3**

MethylcelluloseStockSolution

HSC001

1.4mL

2.1mL

MouseMethylcelluloseBaseMedia

HSC006

2.7mL

3.6mL

MouseMethylcelluloseCompleteMedia

HSC007

3.0mL

4.0mL

MouseMethylcelluloseCompleteMediawithoutEpo

HSC008

3.0mL

4.0mL

*VolumeforDuplicateExperiments

**VolumeforTriplicateExperiments

Procedure

Usesteriletechnique.Useserologicalpipettestotransferandremovesolutions.

PreparationofMouseBoneMarrowCells

Note:

Whenhandlingbiohazardousmaterialssuchassharpneedles,safelaboratoryproceduresshouldbefollowedandprotectiveclothingshouldbeworn.

1.Prepareasuspensionofmononuclearcellsfrommousebonemarrowusingtraditionalmethods.Bothfemursandtibiaefromonemousetypicallyyield2.0-6.0x107hematopoieticcells.AdetailedprotocolcanbefoundinCurrentProtocolsinImmunology,IsolationofMurineMacrophages（1994）Coligan,J.E.etal.eds.JohnWiley&Sons,Inc.,Volume3,Supplement11,14.1.4.

2.Toremovecellclumpsanddebrisafterharvestingthebonemarrowcells,passthecellsuspensionthrougha70mmnylonstrainer.

3.Afterfiltration,thecellsshouldbeusedassoonaspossible.Or,ifdesired,theredbloodcells（RBC）canberemoved.TolyseRBC,useFlowCytometryMouseLyseBuffer（R&DSystems,Catalog#FC003）accordingtotheinstructions.

4.Washthecellsin50mLcentrifugetubeswithroomtemperatureIMDM/2%FBSbycentrifugingat300xgfor8minutes.Removethesupernatecompletelyandresuspendthecellsin10mLofIMDM/2%FBSbygentlepipetting,togenerateasinglecellsuspension.

Note:

Bonemarrowcellsshouldbeusedassoonaspossibleorfrozenaccordingtothestandardfreezingprotocolusedineachlaboratory.

MethylcelluloseAssay

Figure1

1.Thawaliquotsofmethylcellulose-basedmediumatroomtemperatureforapproximately30minutes.Allowthevialstothawwithoutdisturbance.

2.Duringthethawstep,resuspendthecellsamplein10mLofIMDM/2%FBSorinanappropriatevolumeandcount.

3.CalculatethetotalnumberofcellsneededintheexperimentusingTable2todeterminetherecommendedfinalcellnumberper35mmcultureplate.Transfertheappropriatevolumeofcells（plusaslightexcess）intoanew15mLconicaltube.Centrifugefor8minutesand300xg.

Table2.Determiningtheapproximatecellnumberneededforeach35mmcultureplate.

SampleSource

FinalCellNumber*

StockCellNumber（10xFinal）

BoneMarrow（untreated）

1-5x104

1-5x105

PeripheralBlood

1-3x105

1-3x106

Spleen

1-2x105

1-2x106

FetalLiver

1-5x104

1-5x105

*Finalcellnumberper35mmcultureplate（or1.1mLofmedia）

Note:

Thecellplatingnumberslistedaboveserveasareferenceonly.Optimalcellplatingconcentrationshouldbedeterminedbyeachlaboratoryforeachcelltype.

4.RemovethesupernatantandresuspendthecellsinIMDM/2%FBSortheappropriatemediumtothedesiredcellconcentrationforplating（usually10Xtherecommendedcellnumberrequiredper35mmculturedishlistedintheCellPlatingNumberChart）.

a.WiththeexceptionofusingtheproductHSC001,cellsamplesshouldberesuspendedinIMDM/2%FBS.FortheuseofproductHSC001,cellsamplescanberesuspendedintheselectedmediumperinstructionofeachlaboratory.

b.TheoptimalcellplatingconcentrationshouldbedeterminedintheinitialexperimentbyincludingalowerandhighercellconcentrationthanthecellconcentrationrecommendedintheCellPlatingNumberChart.

5.Thetablebelowprovidestherecommendedvolumesofcellsfromthe10xstockandadditionalculturesupplementsorcytokinestobeaddedtothemethylcellulosealiquots.Thefinalconcentrationofmethylcellulosebeforeaddingcellsshouldbeapproximately1.3%forthevariousmethylcellulose-basedmedia.

Table3.Volumesnecessaryforexperimentsusing35mmcultureplatesinduplicateortriplicate.

HSC001

HSC006

HSC007-HSC008

Usingcellsamplesin

Usingcellsamplesin

Usingcellsamplesin

Duplicates

Triplicates

Duplicates

Triplicates

Duplicates

Triplicates

Methylcellulose-basedMedium

1.4mL

2.1mL

2.7mL

3.6mL

3.0mL

4.0mL

CultureSupplementsorCytokinesNeeded

1.6mL

2.4mL

0.3mL

0.4mL

None*

None*

CellsNeeded

0.3mL

0.45mL

0.3mL

0.4mL

0.3mL

0.4mL

*Additionalculturesupplementsorcytokinesarenotneeded.

6.

Figure2

7.Vigorouslyvortexthevialtothoroughlymixthecellswiththemedia.

8.Waitforapproximately20minutesbeforecontinuingwiththeproceduretoallowairbubblestoescape.

9.Add1.1mLofthefinalcellmixtureinto35mmculturedishusinga3mLsyringefittedwitha16gaugeneedle.Spreadthemediaevenlybygentlyrotatingtheplate.

10.Placetwosampledishesandanuncovereddishcontaining3-4mLofsterilewaterina100mmculturedishandcover.Thesterilewaterdishservestomaintainthehumiditynecessaryforcolonydevelopment.

11.Incubatethecellsfor8-12daysat37°Cand5%CO2.Avoiddisturbingtheplateduringtheincubationperiodtopreventshiftingofthecolonies.

Figure3

ColonyScoring

Scorecoloniesattheendoftheincubationperiod.Identifyandcountindividualcoloniesusinganinvertedmicroscopeandascoringgrid.

∙PreparethescoringgridasdescribedintheScoringGridsection.Thediagramprovidedbelowcanbeusedasatemplatetoreproducethescoringgridona100mmculturedish.Markthegridonanew100mmculturedishbyplacingtheculturedishonthetemplateandtracingthegridwithamarkerorpen.

∙RefertotheCountingCriteriasectionforguidancetoidentifyandcountcolonies.

ScoringGrid

Figure4

CountingCriteria

Coloniesconsistingofatleast30cellsarecounted（ortheminimumcellcountsetbyeachlaboratory）.

ColonyTypes

BFU-E（Burstformingunit-erythroid）

Eachcolonyisdefinedasclusterswithaminimalof30cellsthatcanbeseenfromday7onward.Eachindividualclusterconsistedoftiny,irregularshapedcellsthatmayappearfusedtogether.Eachclusternormallycontains5-8cells,andthesizeoftheclusterissimilartothatofasinglemacrophage.Theclustermayvaryinsizesandcolor.AlargeBFU-Eisusuallybrightredandisdifferentiableevenwithouttheuseofamicroscope.SmallerBFU-Emaynotappearredincolorbutisdistinguishablebasedonthemorphology.

BFU-E（40X）

BFU-E（100X）

BFU-E（200X）

CFU-GM（Colonyformingunit-granulocyte,macrophage）

TheclassificationincludesCFU-G（colonyformingunit-granulocyte）CFU-M（colonyformingunit-macrophage）,andCFU-GM（colonyformingunit-granulocyte,macrophage）.Clonogenicprogenitorsofmacrophageswillgiverisetoahomogenouspopulationofmacrophagesthatarecolorless,large,andround.Clonogenicprogenitorsofgranulocyteswillgiverisetoahomogenouspopulationofgranulocytesthatarecolorlessandsmallerthanthatofmacrophages.Theclonogenicprogenitorsorgranulocyte,macrophagewillgiverisetoaheterogenouspopulationofmacrophagesandgranulocytes.Thesedevelopedcoloniesarecolorlessandconsistedofroundcells（granulocytes）andovalcells（macrophages）.Eachindividualcellcanbedisti