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.
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