Materials and Reagents
10-cm tissue culture dishes
sterile PBS x1 -Ca -Mg
DMEM medium
Fetal Bovine Serum
Penicilin/streptomycin x100
sodium pyruvate x100
Trypsin solution
15 ml tubes
Complete DMEM medium (see recipes)
Procedure
pre-warm complete medium, PBS, trypsin to 37°C.
prepare 15ml tube with 4-5ml complete DMEM
prepare 10cm dish with 10ml medium.
Remove medium from culture by vaccum suction.
Wash cells with 2-4ml of PBS.
Add 1ml trypsin. Make sure it covers the entire plate.
Incubate 1-3min at incubator
Once cells begin to detach, collect cells with 1000μl tip (wash cperform up&down with pipetor to collect all cells from plate). add cells to the 15ml tube.
take out 1ml of from the tube and use it to wash the plate and collect remaining cells.
Take proper amount of cells to the plate (e.g. 1/5 volume for 1:5 split, 1/10 volume for 1:10 split etc...)
Put dish with cells in incubator.
Recipes
complete DMEM:
10% FBS, 1:100 Pen/Strep, 1:100 Na-pyruvate