中国财产规划与管理研究会_
来自 : 蚂蚁淘
PREPARINGCELLS
- BringupHUVECandfibroblastsinM199/10%FBS/Pen-Strep(1:100)1-2daysbeforebeADIng.
- SwitchmediumtoEGM-2(Clonetics)thedaybeforebeadingforHUVECandthedaybeforeembeddingforfibroblasts.
- Aconcentrationof~400HUVECperbeadisneeded.
- 20,000fibroblastsperwellisneeded.
COATINGTHEBEADSWITHHUVEC-DAY-1
- TrypsinizeHUVEC.
- Allowbeadstosettle(DONOTCENTRIFUGE!).Aspiratethesupernatantandwashthebeadsbrieflyin1mLofwarmEGM-2medium.
- Mix2500beadsw/1X106HUVECin1.5mLofwarmEGM-2mediuminaFACStube.Placeitverticallyintheincubator.(Thiswillbeenoughfor~10wells.Scaleupifneeded)
- Incubatefor4hoursat37°C,shakingthetubeevery20min.(Goodcoatingiscrucialforsprouting.)
- After4hours,transferthecoatedbeadstoaT25flaskin5mLofEGM-2andleaveO/N.
EMBEDDINGCOATEDBEADSINFIBRINGEL-DAY0
- Preparethe2.0mg/mLfibrinogensolution(Seerecipesection).
- Add0.15Units/mLofaprotinintothefibrinogensolution.
- Transfercoatedbeadstoa15mLconicaltubeandletbeadssettle.ResUSPendbeadsin1mLofEGM-2andtransfertoa1.5mLcentrifugetube.
- Washthebeads3Xwith1mLofEGM-2bypipetingupanddownSLOWLY.
- Countbeadsonacoverslipandresuspendinfibrinogensolutionataconcentrationof~500beads/mL.
- Add0.625Units/mLofthrombintoeachwell.
- Add0.5mLofthefibrinogen/beadsuspensiontoeachwellofa24-wellplate.ChangethePipettetipforeachwell!!!
- Mixthethrombinandthefibrinogenbygoingupanddowngentlywiththepipettetip~4to5times.Becarefulnottomakelargebubbles.
- Leavetheplatefor5mininthehood,thenplaceitinthe37°C-incubatorfor10-15mintogenerateaclot.
- Whilewaitingfortheclot,trypsinizefibroblasts.
- Add1mLofEGM-2perwelldropwise.
- Seedfibroblastsontopoffibringelataconcentrationof20,000cellsperwell.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
