Purpose:

Thismethodisadvantageousforsavingtheoccasionalculturesthatbecomecontaminated.Yeastcontaminatedcultureswillappearcloudywhenslightlyshakenandlymphocyteswillnotclustertogetherasmuchasnormal.IfculturesaresUSPect,adropofculturecanbestreakedonaYPDmediaplatetocheckforgrowthofyeastcolonies,ora5mlsamplecanbetakentoBarnesDiagnosticCenterforidentificationofyeaststrain.

Procedure:

1. Pipet5mlhistopaqueintoa15mlcentrifugetube.

2. Carefullylayerontopofhistopaque10mlofcontaminatedculture(orconcentratedcells/yeastresuspendedingrowthmedia).

3. Centrifugetubefor25minutesat2500rpm(nobrake)atroomtemperature,usingtheTJ-6centrifuge.

4. TheyeastcellswillpellettothebottomofthehistopaquegrADIentandthelymphoblastcellswillbelocatedontopofhistopaquegradient.Removethelymphoblastcellswitha10mldisposablepipet,andtransfertoa15mlcentrifugetube.

5. Washcellsbyadding10mlofwashmediatocells.Centrifuge10minutesat1200RPM,nobrake,atroomtemperature.AspirateoffthewashmediaandresuspendinRPMI-growthmediacontaining1Xantimycotic/antibiotic.Thiswillremovetherestoftheyeastcells.

6. Transferthecellstoa25cm2TissueCultureFlaskandfeedtheculturetwiceaweekwith1Xantimycotic/antibioticmediauntilalltracesofcontaminationaregone.Thiswilldependontheseverityofthecontamination(usuallyforculturesmoderatelycontaminated,2weeksor4feedingswillsuffice).AftercontaminationisnolongervisIBLe,feedthecultureswithgrowthmediacontainingonlyantibioticandnottheantimycotic.

Solutions:

• Washmedia:(1liter)

   To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add: 10.0 ml 2.5 M (100X) Hepes buffer 1.2 ml 50 mg/ml gentamicin reagent Filter sterilize through a 0.22 祄 cellulose acetate filter and store up to 2 weeks at 4 degrees C.

• Growthmedia:(1liter)

  To1literofstrerileRPMI1640with2mML-glutamine,add:165.0mlfetalbovineserum,heatinactivatedat50-60degreesCforonehalfhour
  12.0ml200mM(100X)L-glutamine1.2ml50mg/mlgentamicinreagentFiltersterilizethrougha0.22祄celluloseacetatefilterandstoreupto2weeksat4degreesC.

• 1XCyclosporinmedia:(100ml)

   To 100 ml of growth media add: 1.0 ml 100X cyclosporin A 

• 100XCyclosporinA:(100ml)

   Dissolve 1 mg CSA in 0.1 ml ethanol in a sterile 15 ml centrifuge tube with a small magnetic stirrer. Add 0.02 ml (or 20祃) of Tween 80 and mix well. While continually stirring, add 1 ml RPMI drop by drop. Quanitate to a final volume of 100 ml with RPMI. Filter sterilize with a 0.22 祄 filter. Store at 4 degrees C for up to 4 months.

• Antimycotic/antibioticmedia:

   To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add: 165.0 ml fetal bovine serum, heat inactivated 12.0 ml 200mM (100X) L-glutamine 12.0 ml antimycotic/antibiotic (100X), liquid, Gibco,Cat.No.600-5240AGFiltersterilizethrougha0.22祄celluloseacetatefilterandstoreupto2weeksat4degreesC.

  References:

  Biotechniques,Overhauser,Joan,etal.DepartmentofBiochemistryandMolecularBIOLOGy,ThomasJeffersonUniversity,Philadelphia,PA.

  ================  蚂蚁淘在线  ================

  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：未经蚂蚁淘在线授权不得转载、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：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。