Epicult plus

The Trp53^tm1BrdBrca1^{tm1Aash/F22-24} Tg (BLG-cre) 74Acl/J mouse strain was purchased from The Jackson Laboratory (012620). All mice were maintained in a temperature-controlled room (21 °C) with a 12-h light-dark cycle with 40–60% humidity. Mammary glands from 6-week-old female mice were dissected out as organoids (around 1 cm³) and were cryopreserved in 10% DMSO/FBS in liquid nitrogen for long-term storage. Upon thawing, cryopreserved mammary organoids isolated from 6-week old mice were cut into small pieces and dissociated into single-cell suspensions with the gentleMACS dissociator (Miltenyi Biotec). After single-cell suspension was obtained, cells were cultured with EpiCult Plus (STEMCELL Technologies) on collagen-coated plates (Corning) and the medium was refreshed every other day. An aliquot of 5500 mammary cells was mixed with 50 μl Matrigel (Invitrogen) and 40 μl of the mixture was dispensed in the center of a well of a warm 24-well plate. The cell and Matrigel mixture solidified for 15 min at 37 °C. One ml of mouse mammary organoid growth medium (STEMCELL Technologies) was added to the well. Medium was refreshed 3 times a week.

Highly purified MECs were isolated from cryopreserved mammary glands from 6-week-old mice. Briefly, this process involved dissociating viably cryopreserved preparations into single-cell suspensions. Cryopreserved mammary glands (around 1 cm³) were cut into small pieces for better tissue dissociation, which was performed with gentleMACS (Miltenyi Biotec) following the manufacturer’s protocols. After obtaining a single-cell suspension, cells were cultured with EpiCult Plus (STEMCELL Technologies) on collagen-coated plates (Corning) and the medium was refreshed every other day. For CFC assays, MECs were seeded at single-cell density (100 cells per 24 wells) in collagen-coated plates for 5 days, and LPS at different doses was used to activate NF-κB in MEC cultures.