Purpose:

Celllinesarereactivatedandgrowntoacountof1x108cells.Thecellsarepelletedandstoredfrozenat-80degreesCpriortoDNAextraction.

Timerequired:

15-20minutestobegingrowing2-4cryovials

Procedure:

1. Frozencellsshouldbethawedquickly.Removethecryovialfromitslongtermstoragecontainerinthe-135degreesCCryostar,andplaceimmediatelyina37degreesCwaterbathfor2minutes.

2. Removethecellsfromthevialandplacein10mlwashmedia.Thisisnecessarytoremovetracesofdimethylsulfoxidefromthecells.

3. Centrifugecellsfor10minutesat1200rpm(nobrake)atroomtemperatureusingtheTJ-6centrifuge.

4. Removethesupernatantabovethecellpellet.

5. ResUSPendthecellpelletin7-10mlof1XCyclosporinAmedia.

6. Aspiratehalfofculturemediawithin3-4days.Addgrowthmediaandslightlyincreasevolumby5ml.Increasethevolumeofmediaby5-10mltwotimesaweekbyaspiratingoffhalfofmediafromcultureflask(donottosuctionoffcellsfrombottomofflask)andreplacingitwithfreshgrowthmedia.CellscanbeharvestedforextractionwhenaT-75cm2flaskreachesavolumeof100mlofmediaandthereisamonolayerofcellsonbottomofflask.

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)

   To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add:
  165.0mlfetalbovineserum,heatinactivatedat50-60癈foronehalfhour12.0ml200mM(100X)L-glutamine1.2ml50mg/mlgentamicinreagenFiltersterilizethrougha0.22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 (= 20 祃 ) of Tween 80 and mix well. While continually stirring, add 1 ml RPMI drop by drop. Bring to a final volume of 100 ml with RPMI. Filter sterilize witha 0.22 祄 filter. Store at 4 degrees C for up to 4 months.

  References:

  Dr.RichardTodd,PsychiatryDepartment,ClinicalScienceResearchBuilding.

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