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Splitting cell culture

Author: Gal Haimovich, updated date: , view: 8, Q&A: 0
Tags: Cell Culture and Adherent cells

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

  1. pre-warm complete medium, PBS, trypsin to 37°C.

  2. prepare 15ml tube with 4-5ml complete DMEM

  3. prepare 10cm dish with 10ml medium.

  4. Remove medium from culture by vaccum suction.

  5. Wash cells with 2-4ml of PBS.

  6. Add 1ml trypsin. Make sure it covers the entire plate.

  7. Incubate 1-3min at incubator

  8. 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.

  9. take out 1ml of from the tube and use it to wash the plate and collect remaining cells.

  10. 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...)

  11. Put dish with cells in incubator.



Recipes

complete DMEM:

10% FBS, 1:100 Pen/Strep, 1:100 Na-pyruvate

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