最近有两位战友发信询问我的荧光微球吞噬实验的Protocol。
现分享于此:
LatexBeadsPhagocytosisassay
---Materials
*Fluorescencelabeledlatexbeads(1umdiameter),2.5%aqueoussUSPension
*3%BSAcontaining25mMNa2HPO4,pH6.0
*0,3%(w/v)azide
*Culturemediumcontaining5%FBS
*Distilledwater,PBS
*Bathsonication,6(12)-wellplates
---Cellcultureandtreatment
1,Inoculateplateswith7,0104cells/cm2perwell.Incubateat37℃,5%CO2for24hr,bestuntil50-70%confluenceisreached
2,Removeculturemediumandexposethecellstotestmaterial.Incubateat37℃,5%CO2for24hr.
---Preparationofcoatedlatexbeads
1,Washlatexbeadswithdistilledwaterandpelletat10,000gfor8minatRT.
2,Resuspendlatexbeadsin3%BSAcontaining25mMNa3PO4(pH6.0)andincubateatRTfor15minwithbathsonication.
*CoatingbeadsinBSAinsuresbeadsremaininamonodispersestate.
3,Washthebeadsoncewithculturemediumcontaining5%FBS.
4,Resuspendthebeadsinculturemediumatconcentration2.0%.Thisisbeadsstock.Storedindarknessat4℃.
---Assay
1,Controlsandsamples:Intactcontrol(Nostaining)1well
-Negativecontrol(azidetreated)1well
-Normalcontrol1well
-sample5wells
*Inordertodifferentiatebetweenphagocytosedbeadsandbeadsnonspecificallyadheretothecellsurface,controlcellsareexposedto0,3%(w/v)azidefor10minpriortotheadditionofcoatedbeads.Thistreatmentcompromisesmicroglialenergeticprocessesandfewbeadswereinternalizedasobservedbyfluorescentmicroscopy.Meanfluorescenceofazide-treatedmicrogliawasusedasthenegativecontrolandwassubtractedfromvaluesobtainedinexperimentalsamples.
2,Forexperimentsusing6well-plates,15μlbeadsstockin1mlculturemediumisappliedtoeachwell.Votexthebeadsstockwellandtakeout105μlandaddinto7mlculturemedium.Bathsonificatefor10minatRTindarkness.Thisisbeadsworkingsolution.
Forexperimentsusing12well-plates,6μlbeadsstockin0.4mlculturemediumisappliedtoeachwell.
3,WasheachwelltwicewithPBSandreplacewithbeadsworkingsolution,1ml/wellfor6-wellplate,0.4ml/wellfor12well-plate.Incubateinthedarkat37℃for80-120min.
4,Removebeadsworkingsolutionandwash3timeswithPBStoremoveexcessbeads.
5,LiftthecellsbyscrappingortrypsinizationandwashthecellswishPBS.
6,StainwithPI(4ug/mlfinalconcentration)andrunforFACS.
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