Lentivirus Infection Protocol for stable cell line development ...

This protocol is for the stable cell line construction based on puromycin selection. Day 1: Seed target cells in 24-well plates. The number of seeding cells ... 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Day1:Seedtargetcellsin24-wellplates.Thenumberofseedingcellsdiffersaccordingtothecellproliferationrate. Day2:Targetcellsshouldbeapproximately50%-70%confluent.Forpolybreneaccessiblecells,mixtheculturemediumwithproperconcentrationsofpolybrene.Replacethemediumcompletelywith0.5mlpolybrene-containingmedium.Forpolybrenesensitivecells,thisstepcanbeskipped. Beforeinfection,virusshouldbemeltedonicegentlyandresuspendedinculturemedium.Removetheprecedingmediumandaddlentivirus-containingmediumwith1/2volumeofnormalculturevolume.Culturefor4hoursat37℃,andsupplementfreshmediumtonormalvolume.Therecommendedmediumvolumeoflentivirusinfectionisdisplayedinthefollowingtable4. Table4.Mediumvolumeoflentivirusinfection(1/2volumeforlentivirusinfection) Culturedishtype Surfacearea normalvolumeforcellculture 1/2volumeforlentivirusinfection 96-well 0.3cm2 100ul 50ul 24-well 2cm2 500ul 250ul 12-well 4cm2 1ml 500ul 6-well 10cm2 2ml 1ml Note: 1.Polybreneconcentration Thoughpolybreneincreasestheefficiencyofviralinfection,itistoxictosomecelllines,andthesensitivityvariesfromdifferentcelllines.ForpolybreneaccessiblecellsWerecommendtheworkingconcentrationofpolybreneas6-8μg/ml. 2.OptimalMOIdetectionforcellinfection MOI(multiplicityofinfection)referstothenumberofinfectedviralparticlespercell.ForactivelydividingcellssuchasHeLaor293cells,over80%ofthecellscanexpresstargetgeneswithMOIof1-3.Forthenon-dividingcellslikeprimarycellswithalowinfectionefficiency,werecommendtestingarangeofMOIstodeterminetheoptimalMOIforinfectionandgeneexpressionintargetcelllines.ThelentiviralMOIsofsomecommoncelllineareshowninthetable5. Table5.ThelentiviralMOIsofcommoncellline. Cellline MOIrange Auxiliaryinfectionreagentpolybrene(need/no) K562 20～40 Need Jurkat 50～80 no kasumi 10～30 no NB4 50～80 no U937 20～40 Need THP-1 50～80 Need GBC-SD 30～50 no H929 100～150 no H1299 1～3 Need 95D 2～4 Need A549 20～40 Need SPC-A-1 100～150 Need 7402 10～15 Need Hep3B 10～30 Need HepG2 10～30 Need SMMC-7721 10～30 Need Huh-7 10～30 Need Hela 10～30 Need HOS 20～40 Need Hep-2 10～30 Need HL-60 >100 Need HT-29 10~30 Need PKO 2~4 Need SW480 10～30 Need DLD-1 10～30 Need SK-OV-3 2～4 Need SHG-44 10～30 Need U251 1～3 Need U87 1～3 Need 293T 1～3 Need HUVEC-2C 10~30 Need PC-3 20～40 Need MDA-MB-231 10～30 Need MCF-7 20～40 no Tca8113 10～30 Need RPE 10～30 Need AGS 100～150 Need BGC-823 100～150 Need SGC-7901 10~30 Need MKN-28 20～40 Need MKN-45 20～40 Need BxPc-3 20～40 Need CFPAC-1 50~80 Need Panc-1 2～4 Need HEC-1-B 2～4 Need NIH-3T3 20~40 Need Raw264.7 10～30 no CHO 20～40 Need HSC-T6 10~30 no C6 >100 Need NRK 10～30 Need Note:Influencedbycellsource,algebraandcellstate,theMOIvaluesindifferentlaboratoriesmaybedifferenttosomeextent.Datainthistableareobtainedonconditionthatthecellisingoodstate,andinfectionefficiencycanreach85-100%cells. Day3:Refreshtheculturemedium24hourspostinfection. Day4:Changetofreshmediumwithpuromycin48hourspostinfection.Therecommendedconcentrationofpuromycinrangesfrom1to10μg/mlaccordingtocelllines.Settheuninfectedwild-typecellsascontrolgroupandaddequalvolumeandconcentrationofpuromycin.Replacewithfreshpuromycin-containingmediumevery2or3daysuntilthecontrolgroupcellsdieout.Thenchooseoneofthefollowingstepsaccordingtoexperimentalrequirements. 1.Non-selectingmonoclonalcells Passagetheinfectedcellsandselectwithpuromycinconstantly.Freezethecellmixtureincontinuousthreepassages.Consideringtheheterogeneity,werecommendselectingmonoclonalcellforaconfirmedphenotype. 2.Selectingmonoclonalcells Selectatleastfivemonoclonalcellsafterinfectionandpuromycinselection,andpropagateinpuromycin-containingmedium.DetectiontheexpressionoftargetgenesusingwesternblotorqPCR.Choosethestablecelllinewithproperexpressionleveloftargetgenestopassagethreegenerationsandfreezethestablecellline. Notesforinfectionofspecialcelllines. 1.Suspensioncells Werecommendusingflatfilletcentrifugingtransfectiontoinfectsuspensioncellsorsemi-suspensioncells.Addvirussuspensionintocellculturedish,sealingtightly,andcentrifugeatlowspeedof200×gfor1hourintheflatfilletcentrifuge.Placecellsincellcultureincubatoraftercentrifugingtransfection.Iftheflatfilletcentrifugeisinaccessible,youcansuspendthecellsandtransfercellsintocentrifugetubes,followedbylow-speedcentrifuge,anddiscardthemostofsupernatant.Addvirussuspensionintothetubes,resuspendingcells,placeitatroomtemperaturefor15min(nomorethan30min),andtransferthecellsandvirussuspensionintoplatetoculture.Replacewithfreshculturemediumthenextday. 2.Cellsdifficulttoinfect Forcellsdifficulttoinfect,likeDCcells,werecommendrepeatedinfections.Replacewithfreshvirussuspension24hoursafterthefirstinfection.Repeatedinfectionscanincreasetheinfectionefficiencymarkedly. 3.Non-dividingprimarycells Werecommendhigh-titeradenovirustoinfectthesecellslikeBMSC. DetailedinformationofLentivirusprotocolcanbeseeninLentivirusProtocol. GENEMEDI 6thFloor,BuildingNo.2,KangxinRoad3377,Shanghai,China Email:[email protected][email protected] Telephone:+86-21-50478399   Fax:86-21-50478399 PrivacyPolicy TECHNICALSUPPORT ProductFAQs Technicalmanuals Publications Email:[email protected] GETINTOUCH Facebook LinkedIn Twitter ChineseWebsite Aboutus WithstrongexpertiseinGene&Biogiticsdiscovery,artificialdesign,andState-of-the-ArtManufacturing,GeneMedihelpsacceleratemultiplemodalitiesofMacro-MoleculeDiscovery&Development(M-MD&D),andscalablemanufacturingincludingantibodies,recombinantprotein,andmultipletypesofgenetherapeuticvectorsanddeliveryvehicles(AAV,VLP,etc.). PrivacyPolicy ChineseWebsite Copyright©2019.GenemeidAllrightsreserved. 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