Passaging A1 cell: Difference between revisions

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<h4>For passaging 3 flasks of A1 cells:</h4>
<li>1 flask into 3 flasks and
<li>2 flasks into 2 x 10 cm plates (for making myotubes)


<h3>Materials :</h3>
<li>HS AMEM (see additional protocol for recipe)
<li>APBS ( 100 mls PBS + 25 mls water )
<li>50 ml conical tube of 0.75% gelatin ( warmed to 37 degrees C )
<li>0.05% Trypsin/EDTA  ( 36 ml APBS + 4 ml 10x TE )
<li>1x unplugged pasteur pipettes
<li>20 ml pipettes
<li>10 ml pipettes
<li>3 large flasks-162 cm2
<li>2  10 cm plates
<li>3 conical tubes, 15 ml
<li>1 scalpel no. 10
<h3>Execution:</h3>
Prepare plates and flasks
# 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)
# --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
# aspirate  media off of the flask containing the cells
# Rinse  each black cap flask with 7 mls APBS
# aspirate off the APBS
# take 6 ml TE and put in flask
# wait until almost all cells come off
# stop with 3 mls HS AMEM in each flask
# mix with a pipette using the flow from pipette to detach any adherent cells
# put the cells into the 15 ml conical tube
# centrifuge ( spin 1000 rpm for 3 minutes )
# while you are waiting put 25 mls HS AMEM into each new flask and 10 mls into each 10 cm plate
# aspirate off the liquid in each conical tube
# 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
# check cells  in the microscope
# label with cell type, passage number , date and name
put cells in 25 °c and 2 % CO2 incubator

Revision as of 07:55, 6 August 2014

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