MCF-7 Culturing Protocol:
- Use Eagle’s MEM, supplemented with 10% FBS, 1% penicillin/streptomycin. Can also add non essential amino acids (0.1 mM), Insulin (10ug/mL) and Sodium pyruvate (1mM). Add 10nM estrogen to media for a 3-4x increase in cell numbers. Maintain temperature at 37°C in humidified, concentrated CO2 (5%) atmosphere.
- Once MCF-7 cells reach approximately 90% confluence on plates, remove media and passage cells by rinsing with 1xPBS twice.
- Add 2-3 mL of warm (37°C) 0.25% Trypsin- 0.53 mM EDTA solution to cells to disperse cell layer. Observe under an inverted microscope. Dispersal should happen between 5 and 15 minutes. If cells are not detaching properly, place flask back in 37°C incubation chamber. Do not incubate for more than 3 minutes or so. Note: Do not agitate the chills during dispersal, either by hitting or shaking the flask. This may cause clumping as the cells detach.
- Once MCF-7 cell layer is dispersed (3min at 37°C) deactivate Trypsin by adding 5 ml cells/Trypsin-EDTA to 10mL of complete growth medium (see step 1) in sterile tube. Aspirate cells by gently pipetting
- Centrifuge cells in growth medium for 5 minutes at 125x g-force.
- Remove trypsin/growth medium suspension from tube.
- Resuspend pellet (MCF-7 cells) in 10 mL fresh growth medium (see step 1)
- Plate 1 mL of suspension to each new plate containing 9 mL original growth medium (see step 1), and incubate at 37°C in humidified 5% CO2 atmosphere.
A 1:3 or 1:6 subcultivation ratio is reccommended