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PetakaG3 TMBasicsMaximum volume of media permittedin Petaka isMaximumvolume20 mlMedia injection and withdrawalshould always be done holdingPetaka in a VERTICAL POSITION(more will alter Petaka’s performance!)PetakaG3 TMReady to hostPetakas (RTHP)PREPARING “READY TOHOST” PETAKASHold the Petaka in a vertical position!1. Inject 0.5 ml of Antibiotic solution2. Pump 16 ml of MediumTotal16.5mlKeep 3.5 mlfree to addcells and serumlater3. Keep these Petakas refrigerated at4ºC in a vertical position in standsor in a plastic bag (zip bag)Dehydration and pH should becontrolled if RTHP are kept Inrefrigerator for months!SEEDING CELLS IN “READYTO HOST” PETAKASHolding Petaka a in vertical position1. Disinfect port with 90% Ethanol &a flame2. Warm the Petakas in an incubator,at 37ºC for 20 -30 min in aVERTICAL position3. Inject 1.5ml of serum (*)4. Inject up to 2ml of cell suspension5. Whilst holding the Petaka in avertical position, squeeze it untilthe level of media reaches the airexit & then release until the air inputstops. Then the inside/outsidepressure will be balanced6. Shake gently for 10-15 seconds7. Place in a stand & allow to incubatein a HORIZONTAL positionCulturing cells in mediasupplemented mediaPetakaG3 TM mlSEEDING THE CELLSHolding Petaka in a vertical position!1. Inject 1 ml of Serum for 5%concentration2. Inject 0.5 ml of Antibiotic solution3. Inject X ml of Cell suspension4. Inject (18.5 –X) ml of Medium20Total5. Whilst holding the Petaka in avertical position, squeeze it untilthe level of media reaches the airexit & then release until the air inputstops. Then the inside/outsidepressure will be balanced.6. Incubate Petaka according tothe Cell type (see INCUBATIONPOSITIONS)
PetakaG3 TMHarvestinganchored cellsADHERENT CELLS WITHTRYPSIN-EDTA1. Disinfect the port with 90% Ethanol& a flame2. Completely extract all the mediafrom the Petaka3.Inject 4 ml of 0.25% Tyrpsine-EDTAsolution4. Shake thoroughly5. Incubate at 37ºC for 3 min in ahorizontal position (both sides)6. Check Cell detachment under themicroscope7. WHEN CELLS ARE DETACHED!Tap the Petaka 2-3 times8. Pellet the cells using a Petakacentrifuge (see centrifugation)9. To pellet the cells in the bottomright hand corner of Petaka,position it with the port being theupper most corner of the device10. Introduce Petaka into thecentrifuge rotor pocket11. Set desired speed & time & begincentrifuge12. After centrifuge, the cell pellet willbe visible in the bottom right handcorner13. To extract the pellet, use a micropipette with a 1mm tip14. Transfer the cells to a conical tubePetakaG3 TMHarvesting freesuspended cellsNON ADHERENT CELLS1. Disinfect port with 90% Ethanol & aflame2. Tap the Petaka 2-3 times3. Pellet the cells using a Petakacentrifuge (see centrifugation)5. To pellet the cells in the bottom righthand corner of Petaka, position itwith the port being the upper mostcorner of the device6. Introduce Petaka into the centrifugerotor pocket7. Set desired speed & time & begincentrifuge8. After centrifuge, the cell pellet willbe visible in the bottom right handcorner9. To extract the pellet, use a micropipette with a 1mm tip10. Transfer the cells to a conical tubePetakaG3 TMKeeping cells in “invitro dormancy”(only adherent cells)PUTING THE CELLS IN“IN VITRO DORMANCY”1. After 75% confluent cell growth,remove the Petakas from the incubator2. Keep the Petakas at 20-25ºC in avertical position, away from directsunlight, or Infra-Red sources.3. Keep in a Polyspan Shield4. Periodically Check Cell shape underthe microscopeDO NOT CHANGETHE MEDIUM!In-dormancy survival time depends on:a) Cell typeb) Culture Mediac) Type of Buffer (in the media)d) Stability of the environmental variables
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