Protocols_CellCulture_MCF10A Cell culture

MCF10A Cell culture

Source: Bonini Lab, NU Chicago campus

Note : Do not use Phenol-red containing reagents for this cell culture as Phenol-red transforms the cells from cobblestone-like to a more mesenchymal-like state (spindle shaped)

Media: DMEM/F-12 (1:1) 1X (phenol-red free) (Gibco) with 10% FBS, 1XPen-Strep, Insulin, EGF, Hydrocortisone, Cholextorn(?)

Cell dissociation reagent: 0.5% Trypsin-EDTA (10X) Phenol-red free (Gibco)

Freezing media: Complete media+10%DMSO

Trypsinization protocol:

1. Dilute 10X Trypsin to 5X in Mg2+, Ca2+-free PBS (2ml trypsin, 2ml PBS)

2. Add 3ml of 5X Trypsin to T-75, scale accordingly.

3. Incubate at 37C for 10 min or until most cells become rounded. (These cells are hard to detach so optimize time by monitoring cell detachment)

4. Add 7ml media to stop trypsinization. Spin down and resuspend with fresh media.