Isolation of murine splenocytes 大小鼠实验 资讯
来自 : 蚂蚁淘
Inordertostudyspleencells(e.g.lymphocytes,granulocytes,otherimmunecells),ithelpstomakesingle-cellsUSPensionssothatthecellscanbemanipulatedexvivoeasily.Thisprotocolsuggestswaysinwhichyoucandothiswithoutalotofequipmentorexpensivesupplies.Thisprotocolcanalsobeusedtomakecellsuspensionsfromotherlymphoidorgans,suchasthethymusorlymphnodes(seeCurrentProtocolsinImmunology,Unit1.9[1]).
Allmaterialslistedareforusewithonemouse.
Supplies
- 15mlconicaltube
- 60mmpetridish
- 5mlpipet
- 100μmcellstrainer(cansubstituteautoclavedfinenylonmeshforProtocolB)
- 3mLsteriledisposablesyringe,noneedleattached(ProtocolAonly)
- frosted-endglassslides(x2)(ProtocolBonly)
- 50mlconicaltube(ProtocolBonly)
Reagents
- DMEM-10(about20mL)
- 1LDMEM(with4.5g/Lglucose,L-glutamine,sodiumpyruvate;fromMediatech,catalog#10-013-CM)
- 100mLfetalbovine/calfserum
- 10mL100XPSG(penicillinGsodium,streptomycinsulfate,L-glutamine;fromGibco,catalog#10378-016)
- sterilizeusing0.2μmfilter;storeat4°
ACKlysisbuffer(1mL)- 1Ldeionizedwater
- 8.29gNH4Cl
- 1gKHCO3
- 37.2mgNa2-EDTA
- pHsolutionto7.2-7.4;sterilizeusing0.2μmfilter;storeat4°
70%ethanoltrypanbluesolutionEquipment
- scissors
- forceps
- smallplasticorglassbeaker
- dissectionstage(canbestyrofoamshippingboxlidwrappedinaluminumfoil)
- P1000Pipette
- hemacytometer
- phasemicroscope
- centrifuge
ProtocolA
Set-Up
- Cleandissectionstagewith70%ethanol.
- Addethanoltothebeakerandplaceendsofscissorsandforcepsintothebeakertosterilize.
- Add8-10mLofDMEM-10tothepetridish.
- PlacethecellstrainerintothedishwiththeDMEM-10.
Procedure
- Wetfuronleftsideofsacrificedmouseusing70%ethanol.
- Cutoutthespleen.
- Cutawaythefuralongtheleftsideofthemouse,abouthalf-waybetweenthefrontandbacklegs.
- Cutopenthebodycavity.
- Removethespleenusingtheforceps(thespleenisthecolorofakidneybean;itislongerandflatterthanthekidney).
Placethespleenintothecellstrainer.Usingtheplungerendofthesyringe,mashthespleenthroughthecellstrainerintothepetridish.Rinsethecellstrainerwith5mLDMEM-10.Discardthestrainer.Transferthesuspendedcellstoa15mLconical.Spincellsat800xgfor3minutes.Discardsupernatantandresuspendpelletin1mLACKlysisbuffer.IncubateatRTfor5-10minutes.Add9mLDMEM-10andspinasbefore.Discardsupernatantandresuspendpelletin3mLDMEM-10,discardinganydeadcellmass.Countcells(dilute10μLcellsuspensionintrypanblue,andcountwithhemcytometer).ProtocolB
Set-Up
- sameasProcedureB,exceptreplacestep4with:
- Sterilizethefrostedendoftheglassslidesbydippinginorsprayingwithethanol.Takecaretoonlytouchthenon-frostedendswithyourgloves.
Procedure
- sameasProcedureB,exceptreplacesteps3with:
- PlacethespleendirectlyintotheDMEMinthepetridish.
- Homogenizethespleenbetweenthefrostedendsoftheslides.
- Passthehomogenizedspleenthroughthecellstrainer(ornylonmesh)mountedona50mLconical.
- Continuewithstep4ofProcedureA.
- Keepcellsoniceorat4°ifyoudonotplantousethemrightaway.
- Ifsterilityisdesired,performallstepsinalaminarflowculturehood.
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