产品名称： 上皮细胞转染试剂盒 
英文名称：EpiFectagen
货号：0923
品牌：ScienCell
产地：美国

Introduction
ThedeliveryofforeignDNAintoeukaryoticcellsisoneofthemostcommonmolecularbiologytechniques
tostudybiologicalmechanisms.However,unliketransformedcelllines,theefficienttransfectionofprimary
cellscanbeaproblem.EpiFectagenisacationicpolymer-basedtransfectionsystemspecificallydesignedand
optimizedforefficienttransfectionofprimaryepithelialcells.TransfectionwithEpiFectagencanbecarriedout
inthepresenceofantibioticsandserum.Insteadofnormaltwo-daytransfection,anoptimizedone-day
transfectionprocedurecanbeperformedfortime-savingandhighlyreproducibletransfection.1.5mlof
EpiFectagenreagentissufficientforupto100transfectionsperwellin96-wellplate.
Storage/Handling
Uponreceipt,aliquotandstoreEpiFectagenreagentAat-20°C,avoidrepeatedfreezing/thawingcycles.
Oncethawed,storeEpiFectagenreagentAat4°Canduseinamonth.EpiFectagenreagentBcanbekeptat4°C.
QualityControl
EachlotofEpiFectagenisperformancetestedbytransfectingHumanRenalProximalTubularEpithelial
Cells(HRPTEpiCs,Cat.No.4100,ScienCell™)withPromega®ρSV-bata-Galactosidasecontrolvector.Gene
expressionisassayedbyX-galstaining24hoursposttransfection.Typically,30-60%transfectionefficiencycan
beachieved(Figure1).
ProceduresforTransfectingAdherentCellsin96-wellPlate*
A.Preparationofcells
1.Onthedayoftransfection,coat96-wellplatewithpoly-l-lysineat2μg/cm2.Incubateat37°Cfor2-4
hours.Rinsethepoly-l-lysinecoatedwellswithsteriledeionizedH2Otwicebeforeseedingofcells.The
pre-coatingofpoly-l-lysineensuresgoodandevenepithelialcelladhesion.
2.Selectaflaskofepithelialcellswith60-80%confluency,harvestanddilutecellsinEpithelialCulture
Mediumtogiveafinalconcentrationof~1.1×105cells/ml.
B.Transfectioncomplexformation
1.PrepareplasmidDNAinsteriledeionized
H2Otogiveafinalconcentrationof1μg/μl.To
achievesuccessfultransfection,highqualityDNA
withOD260/OD280of1.8orgreateris
recommended.
2.Foreachwell,add0.5μlplasmidDNA,
10.5μlsteriledeionizedH2Oand2μlEpiFectagen
reagentBintoa1.5mlsterileplastictube.Vortex
gentlyandspindownbriefly.Thenadd7μl
EpiFectagenreagentAtomakethetotalvolumeof
thetransfectionmixturetobe20μl,vortexfor5
secondsandspindown.Incubateatroom
temperaturefor20-30min.
C.Incubationofcellswithtransfectionmixture
1
Figure1.HRPTEpiCsexpressingβ-galactosidase
aftertransfectionusingEpiFectagen.
1.Plate180μlofcellsuspension(~1.1×105cells/ml)ineachwelltogive~2×104cellsperwell.
2.Add20μloftransfectionmixturetoeachwell.Mixbygentlyrockingtheplateside-to-side.
3.Culturethecellsfor~24hoursunderstandardconditions.Orperformamediumchangeafter4-
6hours’incubationwithtransfectionmixtures,replacewith200μlfreshculturemedium,andculturefor
additional16-18hours.Generallylongerincubationtimewithtransfectionmixtureresultsinincreased
transfectionefficiencyanddecreasedcellviability.
4.Harvestcells24hoursposttransfectionandassayforgeneexpression.
*Theamountsofcellsandvarioustransfectionreagentsmentionedintheinstructionarerecommendedfor
performingtransfectionin96-wellplate.Fortransfectioninlargersizewells,theamountsofepithelialcells
andtransfectionreagents(DNA,steriledeionizedH2OandEpiFectagenreagentsA&B)shouldbescaledup
accordingtothesurfaceareaofthewells(Table1).
Table1.RecommendedquantitiesofepithelialcellsandEpiFectagenreagentsperwell.
2
CultureVessel
Growth
Area
(cm2/well)
#of
cells
1μg/μl
DNAstock
(μl)
Sterile
DIH2O
(μl)
EpiFectagen
reagentB(μl)
EpiFectagen
reagentA(μl)
EpiCM
(μl)
96-wellplate0.3520,0000.510.527180
48-wellplate0.845,0001.1244.615.9411
24wellplate2.0115,00
02.96011.6401029
12-wellplate4.0230,00
05.712023812057
6-wellplate9.6550,00
013.7288551934937