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%ethanol
• trypanbluesolution

Equipment

• scissors
• forceps
• smallplasticorglassbeaker
• dissectionstage(canbestyrofoamshippingboxlidwrappedinaluminumfoil)
• P1000Pipette
• hemacytometer
• phasemicroscope
• centrifuge

ProtocolA

Set-Up

1. Cleandissectionstagewith70%ethanol.
2. Addethanoltothebeakerandplaceendsofscissorsandforcepsintothebeakertosterilize.
3. Add8-10mLofDMEM-10tothepetridish.
4. PlacethecellstrainerintothedishwiththeDMEM-10.

Procedure

1. Wetfuronleftsideofsacrificedmouseusing70%ethanol.
2. Cutoutthespleen.
   1. Cutawaythefuralongtheleftsideofthemouse,abouthalf-waybetweenthefrontandbacklegs.
   2. Cutopenthebodycavity.
   3. Removethespleenusingtheforceps(thespleenisthecolorofakidneybean;itislongerandflatterthanthekidney).
3. Placethespleenintothecellstrainer.Usingtheplungerendofthesyringe,mashthespleenthroughthecellstrainerintothepetridish.
4. Rinsethecellstrainerwith5mLDMEM-10.Discardthestrainer.
5. Transferthesuspendedcellstoa15mLconical.
6. Spincellsat800xgfor3minutes.
7. Discardsupernatantandresuspendpelletin1mLACKlysisbuffer.IncubateatRTfor5-10minutes.Add9mLDMEM-10andspinasbefore.
8. Discardsupernatantandresuspendpelletin3mLDMEM-10,discardinganydeadcellmass.
9. Countcells(dilute10μLcellsuspensionintrypanblue,andcountwithhemcytometer).

ProtocolB

Set-Up

• sameasProcedureB,exceptreplacestep4with:

1. Sterilizethefrostedendoftheglassslidesbydippinginorsprayingwithethanol.Takecaretoonlytouchthenon-frostedendswithyourgloves.

Procedure

• sameasProcedureB,exceptreplacesteps3with:

1. PlacethespleendirectlyintotheDMEMinthepetridish.
2. Homogenizethespleenbetweenthefrostedendsoftheslides.
3. Passthehomogenizedspleenthroughthecellstrainer(ornylonmesh)mountedona50mLconical.
4. Continuewithstep4ofProcedureA.

• Keepcellsoniceorat4°ifyoudonotplantousethemrightaway.
• Ifsterilityisdesired,performallstepsinalaminarflowculturehood.

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