请使用支持JavaScript的浏览器! 【原创】荧光微球吞噬实验的Protocol_蚂蚁淘商城
【原创】荧光微球吞噬实验的Protocol
2021-07-29
问题描述:

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,Inoculateplateswith7,0104cells/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.

*请输入10-500个字符
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mornsmile
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此3D图是我的结果。所用细胞并非专职吞噬细胞(astrocyte)。随处理剂量增大,可见荧光细胞的比例以及荧光强度逐渐降低。最下面灰色的细胞群是无荧光对照。
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mornsmile
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下图是Dotplot图形特点:感叹号
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mornsmile
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下图是专职吞噬细胞(microglia)对荧光微球的吞噬。可见所给处理对吞噬功能无明显影响。Histogram
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