Passaging A1 cell: Difference between revisions

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<h4>For passaging 3 flasks of A1 cells:</h4>
== Material ==
<li>1 flask into 3 flasks and
* APBS (100 ml PBS + 25 ml ddwater)
<li>2 flasks into 2 x 10 cm plates (for making myotubes)
* 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


Materials : HS AMEM (see additional protocol for recipe)
== Procedure ==
                APBS ( 100 mls PBS + 25 mls water )
# 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)
50 ml conical tube of 0.75% gelatin ( warmed to 37 degrees C )
# aspirate the old media from the cells
0.05% Trypsin/EDTA  ( 36 ml APBS + 4 ml 10x TE )
# wash the cells 1 times with 5 ml APBS
1x unplugged pasteur pipettes
# remove the APBS
                20 ml pipettes
# add 5 ml TE
                10 ml pipettes
# wait till the cells are detached
                3 large flasks-162 cm2
# stop the TE with 5 ml HS – media
2  10 cm plates
# centrifuge at 1000 rpm, 3 min at 4ºC
                3 conical tubes, 15 ml
# aspirate the supernatant
1 scalpel no. 10
# dissolve the pellet into 3 ml fresh media
# fill in the new flasks 25 ml HS for A1 cells
# add into each flask 1 ml of the cell suspention


 
''The cells grow at 25ºC''
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

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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