Description
ProductSheet
Ect1/E6E7(ATCCCRL
2614)
Organism:Homosapiens,human
Tissue:ectocervixcervix
CellType:epithelialHPV16E6/E7transformed
Age:43years
Gender:female
Morphology:epithelial
GrowthProperties:adherent
BatchSpecificInformation
PleasereadthisFIRST
RefertotheCertificateofAnalysisforbatchspecifictestresults.
StorageTemp.
liquidnitrogen
vaporphase
BiosafetyLevel
2
SAFETYPRECAUTION
ATCChighlyrecommendsthatprotectiveglovesandclothingalwaysbeusedandafullfacemaskalwaysbe
wornwhenhandlingfrozenvials.Itisimportanttonotethatsomevialsleakwhensubmersedinliquidnitrogen
andwillslowlyfillwithliquidnitrogen.Uponthawing,theconversionoftheliquidnitrogenbacktoitsgas
phasemayresultinthevesselexplodingorblowingoffitscapwithdangerousforcecreatingflyingdebris.
Unpacking&StorageInstructions
IntendedUse
Thisproductisintendedforresearchuseonly.Itisnot
intendedforanyanimalorhumantherapeuticor
diagnosticuse.
CompleteGrowthMedium
KeratinocyteSerumFreemedium(GIBCOBRL17005
042)with0.1ng/mlhumanrecombinantEGF,0.05
mg/mlbovinepituitaryextract,andadditionalcalcium
chloride44.1mg/L(finalconcentration0.4mM)
CitationofStrain
Ifuseofthiscultureresultsinascientificpublication,it
shouldbecitedinthatmanuscriptinthefollowing
manner:Ect1/E6E7(ATCCCRL2614)
1. Checkallcontainersforleakageorbreakage.
2. Removethefrozencellsfromthedryicepackagingandimmediatelyplacethecellsatatemperature
below130C,preferablyinliquidnitrogenvapor,untilreadyforuse.
HandlingProcedureforFrozenCells
Toinsurethehighestlevelofviability,thawthevialandinitiatethecultureassoonaspossibleuponreceipt.If
uponarrival,continuedstorageofthefrozencultureisnecessary,itshouldbestoredinliquidnitrogenvapor
phaseandnotat70C.Storageat70Cwillresultinlossofviability.
1. Thawthevialbygentleagitationina37Cwaterbath.Toreducethepossibilityofcontamination,keep
theOringandcapoutofthewater.Thawingshouldberapid(approximately2minutes).
2. Removethevialfromthewaterbathassoonasthecontentsarethawed,anddecontaminateby
dippinginorsprayingwith70%ethanol.Alloftheoperationsfromthispointonshouldbecarriedout
understrictasepticconditions.
3. Itisrecommendedthatthecryoprotectiveagentberemovedimmediately.Transferthevialcontentsto
acentrifugetubecontaining9.0mLcompleteculturemedium.Centrifugethecellsuspensionat
approximately125xgfor5to10minutes.Discardthesupernatantandresuspendthecellpelletinan
appropriateamountoffreshgrowthmedium.
4. Transferthecellstoanappropriatesizevessel.Itisimportanttoavoidexcessivealkalinityofthe
mediumduringrecoveryofthecells.Itissuggestedthat,priortotheadditionofthevialcontents,the
culturevesselcontainingthegrowthmediumbeplacedintotheincubatorforatleast15minutesto
allowthemediumtoreachitsnormalpH(7.0to7.6).
5. Incubatethecultureat37Cinasuitableincubator.A5%CO2inairatmosphereisrecommendedif
usingthemediumdescribedonthisproductsheet.
HandlingProcedureforFlaskCultures
Theflaskwasseededwithcells(seespecificbatchinformation),grown,andcompletelyfilledwithmediumat
ATCCtopreventlossofcellsduringshipping.
AmericanTypeCultureCollection
POBox1549
Manassas,VA20108USA
www.atcc.org
800.638.6597or703.365.2700
Fax:703.365.2750
Email:[email protected]
Orcontactyourlocaldistributor
Page1of3
1. Uponreceipt,visuallyexaminethecultureformacroscopicevidenceofanymicrobialcontamination.
Usinganinvertedmicroscope(preferablyequippedwithphasecontrastoptics),carefullycheckfor
anyevidenceofmicrobialcontamination.Also,checktodetermineifthemajorityofcellsarestill
attachedtothebottomoftheflaskduringshippingtheculturesaresometimeshandledroughlyand
manyofthecellsoftendetachandbecomesuspendedintheculturemedium(butarestillviable).
2. Ifthecellsarestillattached,asepticallyremoveallbut5to10mLoftheshippingmedium.The
shippingmediumcanbesavedforreuse.Incubatethecellsat37Cina5%CO2inairatmosphere
untiltheyarereadytobesubcultured.
3. Ifthecellsarenotattached,asepticallyremovetheentirecontentsoftheflaskandcentrifugeat
125xgfor5to10minutes.Removeshippingmediumandsave.Resuspendthepelletedcellsin10
mLofthismediumandaddto25cm2flask.Incubateat37Cina5%CO2inairatmosphereuntilcells
arereadytobesubcultured.
SubculturingProcedure
ProductSheet
Ect1/E6E7(ATCCCRL
2614)
PleasereadthisFIRST
StorageTemp.
liquidnitrogen
vaporphase
BiosafetyLevel
2
IntendedUse
Thisproductisintendedforresearchuseonly.Itisnot
intendedforanyanimalorhumantherapeuticor
diagnosticuse.
CompleteGrowthMedium
KeratinocyteSerumFreemedium(GIBCOBRL17005
042)with0.1ng/mlhumanrecombinantEGF,0.05
mg/mlbovinepituitaryextract,andadditionalcalcium
chloride44.1mg/L(finalconcentration0.4mM)
CitationofStrain
Ifuseofthiscultureresultsinascientificpublication,it
shouldbecitedinthatmanuscriptinthefollowing
manner:Ect1/E6E7(ATCCCRL2614)
Volumesusedinthisprotocolarefor75cm2flaskproportionallyreduceorincreaseamountofdissociation
mediumforculturevesselsofothersizes.
Note:Thecellsshouldnotbeallowedtobecomeconfluent,subcultureat60to90%ofconfluence.
1. Removeanddiscardculturemedium.
2. Brieflyrinsethecelllayerwith0.25%(w/v)Trypsin0.03%(w/v)EDTAsolutiontoremovealltraces
ofserumthatcontainstrypsininhibitor.
3. Add2.0to3.0mlofTrypsinEDTAsolutiontoflaskandobservecellsunderaninvertedmicroscope
untilcelllayerisdispersed(usuallywithin5to15minutes).
Note:Toavoidclumpingdonotagitatethecellsbyhittingorshakingtheflaskwhilewaitingforthe
cellstodetach.Cellsthataredifficulttodetachmaybeplacedat37Ctofacilitatedispersal.
4. Neutralizethetrypsinbyadding6.0to8.0mLofa1:1mixtureofDulbecco'smodifiedEagle'smedium
andHam'sF12mediumcontaining10%fetalbovineserum.Aspiratecellsbygentlypipetting.
5. ToremovetrypsinEDTAsolution,transfercellsuspensiontocentrifugetubeandspinat
approximately125xgfor5to10minutes.
6. Discardsupernatantandresuspendcellsinfreshserumfreegrowthmedium.Addappropriate
aliquotsofcellsuspensiontonewculturevessels.
7. Placeculturevesselsinincubatorsat37C.Cellswillnotattachwellfor24hoursaftersubculture.
SubcultivationRatio:1:4to1:6
MediumRenewal:Every2to3days.
Note:FormoreinformationonenzymaticdissociationandsubculturingofcelllinesconsultChapter10in
CultureofAnimalCells,amanualofBasicTechniquebyR.IanFreshney,3rdedition,publishedbyAlan
R.Liss,N.Y.,1994
CryopreservationMedium
A1:1mixtureofDulbecco'smodifiedEagle'smediumandHam'sF12medium,85%fetalbovineserum,10%
DMSO,5%.
CellculturetestedDMSOisavailableasATCCCatalogNo.4X.
Comments
Theendocervicalcelllineexpressescharacteristicsofsimplecolumnarepithelium,whereastheectocervical
andvaginalcelllinesexpresscharacteristicsofstratifiedsquamousnonkeratinizingepithelia.
Withoutstimulation,allthreecelllinesproducemacrophagecolonystimulatingfactor(MCSF),transforming
growthfactorbeta1,interleukin8(IL8),prostaglandinE2,thesecretoryleukoproteinaseinhibitor,andthe
polymericimmunoglobulinreceptor.
Theendocervicalcellline(End1/E6E7),butnottheothers,alsoproducethelymphopoieticcytokinesIL6,IL7,
andconsistentlydetectablelevelsofthechemokineknownas"regulateduponactivation,normalTcell
expressedandsecreted"(RANTES).
Stimulationwithinterferongammaandtumornecrosisfactoralpha(TNFalpha)inducesorsignificantlyup
regulatesexpressionofseveralofthecytokinesandchemokinesaswellasmajorhistocompatibilitycomplex
(MHC)classIIantigensinthelines.
Piliated,butnotnonpiliated,NeisseriagonorrhoeastrainF62variantsactivelyinvadetheseepithelialcelllines.
Invasionofthesecellsbygreenfluorescentproteinexpressinggonococciischaracterizedbycolocalization
ofgonococciwithFactin.
References
AmericanTypeCultureCollection
POBox1549
Manassas,VA20108USA
www.atcc.org
800.638.6597or703.365.2700
Fax:703.365.2750
Email:[email protected]
Orcontactyourlocaldistributor
Page2of3
Referencesandotherinformationrelatingtothisproductareavailableonlineatwww.atcc.org.
BiosafetyLevel:2
Appropriatesafetyproceduresshouldalwaysbeusedwiththismaterial.Laboratorysafetyisdiscussedin
thecurrentpublicationoftheBiosafetyinMicrobiologicalandBiomedicalLaboratoriesfromtheU.S.
DepartmentofHealthandHumanServicesCentersforDiseaseControlandPreventionandNationalInstitutes
forHealth.
ATCCWarranty
ProductSheet
Ect1/E6E7(ATCCCRL
2614)
TheviabilityofATCCproductsiswarrantedfor30daysfromthedateofshipment,andisvalidonlyifthe
productisstoredandculturedaccordingtotheinformationincludedonthisproductinformationsheet.ATCC
liststhemediaformulationthathasbeenfoundtobeeffectiveforthisstrain.Whileother,unspecifiedmedia
mayalsoproducesatisfactoryresults,achangeinmediaortheabsenceofanadditivefromtheATCC
recommendedmediamayaffectrecovery,growthand/orfunctionofthisstrain.Ifanalternativemedium
formulationisused,theATCCwarrantyforviabilityisnolongervalid.
Disclaimers
PleasereadthisFIRST
StorageTemp.
liquidnitrogen
vaporphase
BiosafetyLevel
2
IntendedUse
Thisproductisintendedforresearchuseonly.Itisnot
intendedforanyanimalorhumantherapeuticor
diagnosticuse.
CompleteGrowthMedium
KeratinocyteSerumFreemedium(GIBCOBRL17005
042)with0.1ng/mlhumanrecombinantEGF,0.05
mg/mlbovinepituitaryextract,andadditionalcalcium
chloride44.1mg/L(finalconcentration0.4mM)
CitationofStrain
Ifuseofthiscultureresultsinascientificpublication,it
shouldbecitedinthatmanuscriptinthefollowing
manner:Ect1/E6E7(ATCCCRL2614)
AmericanTypeCultureCollection
POBox1549
Manassas,VA20108USA
www.atcc.org
800.638.6597or703.365.2700
Fax:703.365.2750
Email:[email protected]
Orcontactyourlocaldistributor
Page3of3
Thisproductisintendedforlaboratoryresearchpurposesonly.Itisnotintendedforuseinhumans.
WhileATCCusesreasonableeffortstoincludeaccurateanduptodateinformationonthisproductsheet,
ATCCmakesnowarrantiesorrepresentationsastoitsaccuracy.Citationsfromscientificliteratureand
patentsareprovidedforinformationalpurposesonly.ATCCdoesnotwarrantthatsuchinformationhasbeen
confirmedtobeaccurate.
Thisproductissentwiththeconditionthatyouareresponsibleforitssafestorage,handling,anduse.ATCC
isnotliableforanydamagesorinjuriesarisingfromreceiptand/oruseofthisproduct.Whilereasonableeffort
ismadetoensureauthenticityandreliabilityofstrainsondeposit,ATCCisnotliablefordamagesarisingfrom
themisidentificationormisrepresentationofcultures.
PleaseseetheenclosedMaterialTransferAgreement(MTA)forfurtherdetailsregardingtheuseofthis
product.TheMTAisalsoavailableonourWebsiteatwww.atcc.org
AdditionalinformationonthiscultureisavailableontheATCCwebsiteatwww.atcc.org.
ATCC2014.Allrightsreserved.ATCCisaregisteredtrademarkoftheAmericanTypeCultureCollection.[01/14]
