Dissociating Organoids ( Click to Download )

1. Resuspend one to two tubes of frozen organoids with 10 ml total RMFC media, plate on 10cm or 15cm plate and incubate 1-2 hours at 37C.
2. Collect non-adherent cells with a pipet to a 50-ml conical, wash plate with 10 ml PBS, collect to conical and spin down cells 1000 rpm 10’.
3. Resuspend in 10 ml cold PBS + 0.1 BSA.  Pass organoid solution 8-10X through an 18G needle attached to a 10-ml syringe.  Transfer to 15-ml conical tube.
4. Spin down organoids at 1200 rpm for 5 min; aspirate the supernatant.
5. Resuspend cells in 2 ml 0.05% Trypsin/0.53 mM EDTA; incubate at 37C for 8-10 minutes to break apart the organoids (it helps to pipet up and down first for 1-2 min of the 8-10).
6. Pipet up and down a few times with a p1000 pipet to further break up clumps (cells will have clumped together due to DNA release).
7. Add 8 ml RMFC + DNase to digest aggregated DNA from dead cells (100 ml of 5 mg/ml stock); pipet up and down to break up clusters.
8. Add more media to dilute the suspension.
9. Filter cell/organoid mix through a 40 mm filter into a 50-ml conical tube.
10.  Wash the filter with an additional 8-10 ml RMFC.
11.  Spin down the filtered cells at 1200 rpm for 5 min; aspirate the supernatant.
12.  Resuspend the cells in 10 ml MC media for sorting or plating.

Mammary complete (MC)

(MEBM + Bullet kit)

Purchased from Lonza (Cat# CC-3150)

MEBM basal media +

Bullet kit: Insulin (5 µg/ml), Hydrocortisone (0.5 µg/ml), hEGF (10 ng/ml), Pituitary Extract (52 µg/ml), Gentamycin (optional)

1% Antibiotic/Antimycotic (Invitrogen)

Mammosphere media (MM)

MC media except leave out pituitary extract

Reduction Mammary Fibroblast media (RMFC)

DMEM High glucose (Invitrogen)

10% calf serum

1% Antibiotic/Antimycotic (Invitrogen)