Ultra low attachment surface plate

To examine colony formation ability, transfected cells were seeded in six well-plate for 12 days (1,000 cells were seeded into each well of a six-well plate). Then, the cells were fixed with 4% paraformaldehyde (PFA), stained with 0.2% crystal violet (Beyotime, Shanghai, China) and photographed. In the meantime, cell proliferation was further detected by the CCK8 kit at 24, 48, and 72 h. Experiments were repeated three times independently. For Sphere-forming assays, 500 cells were cultured in 6-well Ultra-low Attachment surface plate (Corning#3,471) with serum-free DMEM medium containing for 2 weeks. Cells were fed with 1 ml of medium every 2 days. Photos were taken using an inverted microscope and the sphere numbers in each well were quantified. GraphPad Prism version 8.0 was used for statistical analyses.

Tissues were collected from women admitted for reduction mammoplasty, who were recruited under an approved IRB protocol (NU15B07). All participants provided written informed consent. Breast tissue to be processed is transferred into a sterile petri dish and chopped into small pieces using a scalpel. The minced tissue was transferred to a sterile 50 ml tube and 30 ml of Kaighn’s Modification media (Gibco #21127022) containing collagenase from Clostridium histolyticum (Sigma Aldrich, #C0130) was added, final collagenase concentration is 1 mg/mL. Media containing collagenase is filtered using a 0.22 μm filter. The Falcon tube is sealed with parafilm and tissue is gently dissociated on a shaker at 100 rpm and 37 °C, overnight (16 h). The following day, organoids are collected by the centrifugation of the suspension at 114 × g for 5 min. The supernatant is discarded, and the organoid pellet washed two-three times with PBS. Organoids with a size between 40 and 100 μm are collected and resuspended in fresh media (3 mL) and added to a six-well plate (Ultra-Low Attachment Surface plate, Corning # CLS3471). Organoids are allowed to stabilize for 24 h before use in the experiments.

Adrenals of 10–15 mice (age 2–5 months, both sexes) per experiment were excised and placed in petri dishes with ice-cold PBS. Fat tissue surrounding the adrenals was carefully removed and the adrenal cortex was thoroughly isolated from the medulla. All the cortical tissues were pooled, pelleted (350×g, 5 min), and digested for 20 min at 37 °C while shaking (1.8 mg/ml of collagenase, 10 mg/ml of BSA, and 0.18 mg/ml of DNase in PBS, all from Sigma–Aldrich). The digestion was stopped by washing twice in PBS and the cells were resuspended in 1 ml of high-glucose Dulbecco's modied Eagle medium (DMEM/F12, Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Biochrom), 1% antibiotic-antimycotic solution (Gibco, Thermo Fisher Scientific), 1% l-glutamine (PAA Laboratories), and 20 ng/ml of basic fibroblast growth factor (bFGF) (Sigma–Aldrich). Isolated cells were cultured in ultra-low-attachment surface plates (Corning) at 37 °C in a humidified atmosphere (95% O₂, 5% CO₂). To induce Cre recombination in vitro in cells isolated from the Nes-CreERT^+/-;Rosa26-eYFP^+/+ mice, 1 μM of 4OH-tamoxifen (Sigma–Aldrich) was added to the culture the first 3 days after isolation. Stimulation of the adrenocortical cells with 4 μg/ml of insulin (Thermo Fisher Scientific) or 1 μg/ml of leptin (Sigma–Aldrich) started on day 2 and continued throughout the entire experiment.

Il-1r1^−/− flox control and knockout mice have been previously described (Abdulaal et al., 2016 (link)) and were provided by A. Waisman (University of Mainz, Mainz, Germany), W. Muller, and E. Pinteaux (The University of Manchester, Manchester, England, UK). Bone marrow cell suspensions were collected from femurs and tibias of 8–15-wk-old mice by flushing with complete DMEM (10% FBS and 1% penicillin/streptomycin solution) using Myjector U-100 insulin syringes with 29G × 0.5 needles. Cell aggregates were resuspended by gentle pipetting, and the solution was passed through a 40-µm nylon web. After centrifugation, cells were resuspended in complete DMEM supplemented with 15% l-929 cell–conditioned medium (as a source of M-CSF) to induce macrophage differentiation. Cells were seeded on 12- or 6-well ultra-low attachment surface plates (Corning) and cultured in a humidified incubator at 37°C and 5% CO₂. At day 7, differentiated macrophages were washed and incubated with complete DMEM (control) or 100 ng/ml LPS (Sigma-Aldrich) and 50 ng/ml IFN-γ (PeproTech) or with 4434 melanoma– or NIH3T3 fibroblast–conditioned supernatant. After 24 h, cells were washed and incubated with complete DMEM for 24 h. Macrophage-conditioned media was collected and analyzed by ELISA to detect mature secreted IL-1β as described in the ELISA section.