Difference between revisions of "Passaging A1 cell"

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<h4>For passaging 3 flasks of A1 cells:</h4>
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== Material ==
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<li>1 flask into 3 flasks and
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* APBS (100 ml PBS + 25 ml ddwater)
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<li>2 flasks into 2 x 10 cm plates (for making myotubes)
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* TE in APBS (36 ml APBS + 4 ml TE)
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* Gelatin (warmed to 37ºC)
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* HS for A1 cells
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* 1x 15 ml FALCON tubes
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* Pipettes
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* 3x 175 cm2 flasks
  
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<h3>Materials :</h3>
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== Procedure ==
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<li>HS AMEM (see additional protocol for recipe)
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# put 10 ml Gelatin into the new 175 cm2 flasks ( transfer the gelatine from the first to the second and from the second into the third → remove the remaining liquid)
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<li>APBS ( 100 mls PBS + 25 mls water )
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# aspirate the old media from the cells
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<li>50 ml conical tube of 0.75% gelatin ( warmed to 37 degrees C )
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# wash the cells 1 times with 5 ml APBS
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<li>0.05% Trypsin/EDTA  ( 36 ml APBS + 4 ml 10x TE )
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# remove the APBS
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<li>1x unplugged pasteur pipettes
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# add 5 ml TE
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<li>20 ml pipettes
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# wait till the cells are detached
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<li>10 ml pipettes
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# stop the TE with 5 ml HS – media
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<li>3 large flasks-162 cm2
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# centrifuge at 1000 rpm, 3 min at 4ºC
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<li>2  10 cm plates
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# aspirate the supernatant
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<li>3 conical tubes, 15 ml
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# dissolve the pellet into 3 ml fresh media
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<li>1 scalpel no. 10
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# fill in the new flasks 25 ml HS for A1 cells
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# add into each flask 1 ml of the cell suspention
  
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''The cells grow at 25ºC''
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<h3>Execution:</h3>
 
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Prepare plates and flasks
 
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# 1 score the 10 cm plates using the scalpel making 0.5 x 0.5 cm square grid across the plate.  (This limits the size of the myotubes to a reasonable size)
 
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# 2 --put 10 mls gelatin into each black cap flask and coat the bottom of the flask
 
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--transfer the gelatin from the first flask into the second and coat, then the same procedure for the third flask
 
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--then coat the 10 cm plates
 
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Passage the cells
 
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# 3 aspirate  media off of the flask containing the cells
 
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# 4 Rinse  each black cap flask with 7 mls APBS
 
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# 5 aspirate off the APBS
 
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# 6 take 6 ml TE and put in flask
 
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# 7 wait until almost all cells come off
 
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# 8 stop with 3 mls HS AMEM in each flask
 
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# 9 mix with a pipette using the flow from pipette to detach
 
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any adherent cells
 
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# 10 put the cells into the 15 ml conical tube
 
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# 11 centrifuge ( spin 1000 rpm for 3 minutes )
 
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# 12 while you are waiting put 25 mls HS AMEM into each new flask
 
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and 10 mls into each 10 cm plate
 
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# 13 aspirate off the liquid in each conical tube
 
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# 14 flasks. use a pipette and resuspend cells in 6 mls HS MEM and put 2 mls of resuspension into each new flask!
 
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10 cm plates: resuspend 2 flasks in 4 mls HS MEM and put 2 mls of resuspension into each 10 cm dish
 
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# 15 check cells  in the microscope
 
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# 16 label with cell type, passage number , date and name
 
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put cells in 25 °c and 2 % CO2 incubator
 

Latest revision as of 14:23, 8 June 2015

Material

  • APBS (100 ml PBS + 25 ml ddwater)
  • TE in APBS (36 ml APBS + 4 ml TE)
  • Gelatin (warmed to 37ºC)
  • HS for A1 cells
  • 1x 15 ml FALCON tubes
  • Pipettes
  • 3x 175 cm2 flasks

Procedure

  1. put 10 ml Gelatin into the new 175 cm2 flasks ( transfer the gelatine from the first to the second and from the second into the third → remove the remaining liquid)
  2. aspirate the old media from the cells
  3. wash the cells 1 times with 5 ml APBS
  4. remove the APBS
  5. add 5 ml TE
  6. wait till the cells are detached
  7. stop the TE with 5 ml HS – media
  8. centrifuge at 1000 rpm, 3 min at 4ºC
  9. aspirate the supernatant
  10. dissolve the pellet into 3 ml fresh media
  11. fill in the new flasks 25 ml HS for A1 cells
  12. add into each flask 1 ml of the cell suspention

The cells grow at 25ºC

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