A1 cell passaging myotube

From Tanaka Wiki
Revision as of 14:23, 8 June 2015 by Stephanh (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

For passaging 3 flasks of A1 cells

  • 1 flask into 3 flasks and
  • 2 flasks into 2 x 10 cm plates (for making myotubes)

Material

  • HS AMEM (see additional protocol for recipe)
  • APBS ( 100 mls PBS + 25 mls water )
  • 50 ml conical tube of 0.75% gelatin ( warmed to 37 degrees C )
  • 0.05% Trypsin/EDTA ( 36 ml APBS + 4 ml 10x TE )
  • 1x unplugged pasteur pipettes
  • 20 ml pipettes
  • 10 ml pipettes
  • 3 large flasks-162 cm2
  • 2 10 cm plates
  • 3 conical tubes, 15 ml
  • 1 scalpel no. 10


Procedure

Prepare plates and flasks

  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)
  2. --put 10 mls gelatin into each black cap flask and coat the bottom of the flask

--transfer the gelatin from the first flask into the second and coat, then the same procedure for the third flask --then coat the 10 cm plates

Passage the cells

  1. aspirate media off of the flask containing the cells
  2. Rinse each black cap flask with 7 mls APBS
  3. aspirate off the APBS
  4. take 6 ml TE and put in flask
  5. wait until almost all cells come off
  6. stop with 3 mls HS AMEM in each flask
  7. mix with a pipette using the flow from pipette to detach any adherent cells
  8. put the cells into the 15 ml conical tube
  9. centrifuge ( spin 1000 rpm for 3 minutes )
  10. while you are waiting put 25 mls HS AMEM into each new flask and 10 mls into each 10 cm plate
  11. aspirate off the liquid in each conical tube
  12. flasks. use a pipette and resuspend cells in 6 mls HS MEM and put 2 mls of resuspension into each new flask! 10 cm plates: resuspend 2 flasks in 4 mls HS MEM and put 2 mls of resuspension into each 10 cm dish
  13. check cells in the microscope
  14. label with cell type, passage number , date and name

put cells in 25 °c and 2 % CO2 incubator