Outline:

TomeasureNKcellkilling,suitabletargetcellsarelabeledwith⁵¹Cr,washedandincubatedtogetherwiththekillercells(andtreatments).Largeamountsoflabelisreleasedintothesupernatantupondisruptionofmembraneintegritybythekillingprocessandcanbemeasuredinagamma-counter.

Supplies&Equipment:

• 37°Cwaterbath(withleadcontainerfor50mltubetoshieldgammarADIation,withinaplasticcontainertopreventleadcontaminationofthewaterbath)
• 50mltubes
• roundbottom96-wellplates
• centrifugefor50mltubesand96-wellplates
• wetice

Reagents:

• ⁵¹Cr(sodiumchromateinsterilesolution,NEZ#030S,DuPontNEN,Wilmington,DE),1µCi/µl
• R10media(=RPMI1640+10%FBS)
• PBS+2%EDTA
• TritonX-100solution(10%indH₂O)

Effectorcellpreparation:

ThemouseNK-celllineKY1isusedasaneffectorcell.Duringrecovery(e.g.afterfreezing),thesecellsrequire1000U/mlIL-2andagraduallyweanedto50U/mlIL-2.Theeveningbeforetheassay(day-1),passagethecellsfrom50U/mlIL-2intothedesiredlevelofIL-2forovernight(16h)stimulation.Occasionally,IL-2levels>50U/mlarenecessarytomaintainkillingactivityofKY1cells.

Targetcellpreparation:

YAC-1cellsaretheoptimaltargetformouseNKcells(KY1).Theygrowveryfastandneedtobepassagedeveryotherday.OnlycellswithhighviABIlity(>95%inthetrypanbluetest)shouldbeused.Otherwise,spontaneousreleaseoflabelwillbehighanddeterminationofspecifickillingwillbedifficult.Passageonday-1.Seed2x10⁶cellsperflask(20ml).

Naturalkillingassay:

• Harvesttargetcells.Gentlehandlingwillassurelowbackground.
• Countandestimateviability(trypanbluetest).
• Adjustcellconcentrationto10⁷cells/mlR10mediaina50mltube..
• Transfertherequiredvolume(willneed~200µlcellsUSPensionper96-wellplate)intofresh50mltube.Theremainingstepsuseradioactivity,obeysafetystandards.Thinkahead,tocutdownexposuretime.Maximizedistanceanduseleadshielding.
• Add50µl(=50µCi)⁵¹Crper200µlcellsuspension.Standardsetupwouldbe:1mlcellsuspension+250µl(=250µCi)⁵¹Cr.
• Incubateinat37°Cfor1h(waterinaleadcontainerinaplasticcontainerinawaterbath).Swirlgentlyevery15min.
• Spinat1000rpmfor5min,removesupernatant.
• Resuspendin40mlprewarmedR10media,incubateagain1hourat37°Cwithrepeatedgentleswirling.Thisallowstheremovaloftheinitiallyhighrateofspontaneousleakageandwillfurtherminimizebackground.
• Preparetreatmentsin100µlvolume,storeonice.Label96-wellplate.
• HarvesteffectorcellsusingPBS+2%EDTA,count.Willneed>10x10⁶effectorcells.Standardsetup:50µleffector(2x10⁵cellsforE:T20:1),50µltarget(1x10⁴cells),100µltreatment.

  E:T	E/50µl	T/50µl	E/ml
  1:1	1x104	104	2x105
  10:1	1x105	104	2x106
  20:1	2x105	104	4x106
  50:1	5x105	104	1x107
  100:1	1x106	104	2x107
  200:1	2x106	104	4x107

• Adjusteffectorcellconcentration
  • to4x10⁶cells/ml(forcommonlyusedE:Tof20:1),aliquot50µlintoeachwell(triplicates),
• After1hourincubation,spintargetcellsat1000rpmfor5min.Decantsupernatant,resuspendin40mlR10media.Spinagainandresuspendin50xtheoriginalcellsuspensionvolume(i.e.in10mlfor200µlcellinitialsuspensionvolume),therebyadjustingtheconcentrationto10⁴/50µl.Commonly5,000to50,000targetcells/wellareused.
• Aliquot50µltoNKcellsin96-wellplates.
• Add100µltreatment.
• Asanegativecontrol(spontaneousreleaseoflabel)aliquot3x50µltargetcellsinto96-wellplate.Add150µlR10media.
• Asapositivecontrol(maximalreleaseoflabelupondetergentorfreeze/thawlysis)aliquot3x50µltargetcellsinto96-wellplate.Add130µlR10media(keepsafedistancetoothersamplestoavoidspillingdetergentbubblestootherwells;bestonseparateplate).
• Labelplatesasbeingradioactive,incubateat37°Cfor4hoursin5%CO₂incubator.Longerincubationmayoccasionallybeused,butnaturalkillingshouldbecompletedwithin4hoursandlongerincubationmayinvolveprocessesotherthanspontaneouskilling(e.g.antibody-mediatedcytotoxicity).
• Aftertheincubation,add20µl10%TritonX-100toeachpositivecontrol.Pipetupanddowntoachievecompletelysis.
• Spin96-wellplatesat500gfor5minutes.
• Carefullypipet100µlofthesupernatantintoindividualtubes.Avoiddisturbingthecellpellet,becauseharvestingofintacttargetswillerroneouslyindicateahigherlevelofkilling.
• Countindividualtubesingammacounter.Expectedrangeisbetween1,000-10,000cpmforpositivecontrol,100-2,000cpm(10-20%)spontaneousreleasefornegativecontrol.
• Calculatespecificcytotoxicity(expectedrateforKY1/YAC-1is~50%):

  %specificcytotoxicity=100x(exp-spont)/(max-spont)

References:

• Kiessling,R.,Klein,E.,andWigzell,H.\"Natural\"killercellsinthemouse.I.CytotoxiccellswithspecificityformouseMoloneyleukemiacells.Specificityanddistributionaccordingtogenotype.Eur.J.Immunol.5(2):112-117,1975.
• Yokoyama,W.M.NaturalkillercellsandtheNKgenecomplex.In:Cellsurfaceandmessengermoleculesoftheimmunesystem,editedbyWeir,D.M.,Herzenberg,L.A.,andBlackwell,C.Cambridge,MA:BlackwellScience,Inc.1996,p.65.1-65.21.