PREPARINGCELLS

1. BringupHUVECandfibroblastsinM199/10%FBS/Pen-Strep(1:100)1-2daysbeforebeADIng.
2. SwitchmediumtoEGM-2(Clonetics)thedaybeforebeadingforHUVECandthedaybeforeembeddingforfibroblasts.
3. Aconcentrationof~400HUVECperbeadisneeded.
4. 20,000fibroblastsperwellisneeded.

COATINGTHEBEADSWITHHUVEC-DAY-1

1. TrypsinizeHUVEC.
2. Allowbeadstosettle(DONOTCENTRIFUGE!).Aspiratethesupernatantandwashthebeadsbrieflyin1mLofwarmEGM-2medium.
3. Mix2500beadsw/1X10⁶HUVECin1.5mLofwarmEGM-2mediuminaFACStube.Placeitverticallyintheincubator.(Thiswillbeenoughfor~10wells.Scaleupifneeded)
4. Incubatefor4hoursat37°C,shakingthetubeevery20min.(Goodcoatingiscrucialforsprouting.)
5. After4hours,transferthecoatedbeadstoaT25flaskin5mLofEGM-2andleaveO/N.

EMBEDDINGCOATEDBEADSINFIBRINGEL-DAY0

1. Preparethe2.0mg/mLfibrinogensolution(Seerecipesection).
2. Add0.15Units/mLofaprotinintothefibrinogensolution.
3. Transfercoatedbeadstoa15mLconicaltubeandletbeadssettle.ResUSPendbeadsin1mLofEGM-2andtransfertoa1.5mLcentrifugetube.
4. Washthebeads3Xwith1mLofEGM-2bypipetingupanddownSLOWLY.
5. Countbeadsonacoverslipandresuspendinfibrinogensolutionataconcentrationof~500beads/mL.
6. Add0.625Units/mLofthrombintoeachwell.
7. Add0.5mLofthefibrinogen/beadsuspensiontoeachwellofa24-wellplate.ChangethePipettetipforeachwell!!!
8. Mixthethrombinandthefibrinogenbygoingupanddowngentlywiththepipettetip~4to5times.Becarefulnottomakelargebubbles.
9. Leavetheplatefor5mininthehood,thenplaceitinthe37°C-incubatorfor10-15mintogenerateaclot.
10. Whilewaitingfortheclot,trypsinizefibroblasts.
11. Add1mLofEGM-2perwelldropwise.
12. Seedfibroblastsontopoffibringelataconcentrationof20,000cellsperwell.

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