Difference between revisions of "Passaging A1 cell"
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1 flask into 3 flasks and
2 flasks into 2 x 10 cm plates (for making myotubes)
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
Line 20: | Line 20: | ||
Prepare plates and flasks | 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) | + | # 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 | + | # 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 | --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 | --then coat the 10 cm plates | ||
Passage the cells | Passage the cells | ||
− | 3 aspirate media off of the flask containing the cells | + | # 3 aspirate media off of the flask containing the cells |
− | 4 Rinse each black cap flask with 7 mls APBS | + | # 4 Rinse each black cap flask with 7 mls APBS |
− | 5 aspirate off the APBS | + | # 5 aspirate off the APBS |
− | 6 take 6 ml TE and put in flask | + | # 6 take 6 ml TE and put in flask |
− | 7 wait until almost all cells come off | + | # 7 wait until almost all cells come off |
− | 8 stop with 3 mls HS AMEM in each flask | + | # 8 stop with 3 mls HS AMEM in each flask |
− | 9 mix with a pipette using the flow from pipette to detach | + | # 9 mix with a pipette using the flow from pipette to detach |
any adherent cells | any adherent cells | ||
− | 10 put the cells into the 15 ml conical tube | + | # 10 put the cells into the 15 ml conical tube |
− | 11 centrifuge ( spin 1000 rpm for 3 minutes ) | + | # 11 centrifuge ( spin 1000 rpm for 3 minutes ) |
− | 12 while you are waiting put 25 mls HS AMEM into each new flask | + | # 12 while you are waiting put 25 mls HS AMEM into each new flask |
and 10 mls into each 10 cm plate | and 10 mls into each 10 cm plate | ||
− | 13 aspirate off the liquid in each conical tube | + | # 13 aspirate off the liquid in each conical tube |
− | 14 flasks. use a pipette and resuspend cells in 6 mls HS MEM and put 2 mls of resuspension into each new flask! | + | # 14 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 | 10 cm plates: resuspend 2 flasks in 4 mls HS MEM and put 2 mls of resuspension into each 10 cm dish | ||
− | 15 check cells in the microscope | + | # 15 check cells in the microscope |
− | 16 label with cell type, passage number , date and name | + | # 16 label with cell type, passage number , date and name |
put cells in 25 °c and 2 % CO2 incubator | put cells in 25 °c and 2 % CO2 incubator |
Revision as of 09:04, 2 July 2014
For passaging 3 flasks of A1 cells:
Materials :
Execution:
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
- 3 aspirate media off of the flask containing the cells
- 4 Rinse each black cap flask with 7 mls APBS
- 5 aspirate off the APBS
- 6 take 6 ml TE and put in flask
- 7 wait until almost all cells come off
- 8 stop with 3 mls HS AMEM in each flask
- 9 mix with a pipette using the flow from pipette to detach
any adherent cells
- 10 put the cells into the 15 ml conical tube
- 11 centrifuge ( spin 1000 rpm for 3 minutes )
- 12 while you are waiting put 25 mls HS AMEM into each new flask
and 10 mls into each 10 cm plate
- 13 aspirate off the liquid in each conical tube
- 14 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
- 15 check cells in the microscope
- 16 label with cell type, passage number , date and name
put cells in 25 °c and 2 % CO2 incubator