Collagenase ia

Connexin cRNAs were synthesized using the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX) according to the manufacturer’s instructions. The amount of cRNA was quantitated by measuring the absorbance at 260 nm.
Adult female Xenopus laevis frogs were anesthetized with tricaine and a partial ovariectomy was performed in accordance with protocols approved by the Animal Care and Use Committee at Rosalind Franklin University in North Chicago, IL. The oocytes were manually defolliculated after treating them with collagenase IA (Worthington Biochemical Corporation, Lakewood, NJ). Stage V and VI oocytes were selected and pressure-injected using a Nanoject variable microinjection apparatus (model No. 3-000-203, Drummond Scientific, Broomal, PA) with 36.8 nl of 0.5–600 ng/μl of connexin cRNA and 5 ng/36.8 nl of oligonucleotides antisense to mRNA for Xenopus Cx38 as previously described (Ebihara 1996 (link)). The oocytes were incubated overnight at 18 °C in L-15 (GIBCO-Invitrogen, Carlsbad, CA) containing 2 mM CaCl₂ prior to performing electrophysiological experiments.

Isolation of the human mammary epithelial cells from surgical specimens were performed as previously reported [32 (link)]. Briefly, human specimens were manually dissociated and digested in DMEM supplemented with 20 mM HEPES (Thermo Fisher Scientific, Waltham, MA, USA), 1 mg/mL collagenase IA, 100U/mL hyaluronidase (both Worthington Biochemical Corporation, Lakewood, NJ, USA), 12 U/mL DNase I (Merck, Darmstadt, Germany), 1% penicillin-streptomycin and 0.25 μg/mL fungizone (both Sigma Aldrich, St. Louis, MO, USA). Initial digests were washed with DMEM, lysed of red blood cells and single-cell suspensions were obtained by further 10 min digest in trypsin (Thermo Fisher Scientific) at room temperature. Cells were filtered through a 70 μm nylon mesh (Corning, Somerville, MA, USA) prior to further analysis or culture.

Total mammary cells were derived as previously described [31 (link)]. Total cell populations were isolated from multiple lesions and pooled from 2–3 mice. Briefly, all mouse mammary glands were dissected, and lymph nodes removed, manually dissociated and then digested in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 mg/mL collagenase IA, 100 U/mL hyaluronidase (Worthington Biochemical Corporation, Lakewood, NJ, USA) and 2% foetal calf serum (FCS) for three h at 37 °C. The freshly isolated total cell preparations were cultured overnight in non-adherent conditions [1:1 volumes of DMEM and Ham’s F12 nutrient mix (Thermo Fisher Scientific), supplemented with 10 ng mL⁻¹ epidermal growth factor (EGF), 20 ng mL⁻¹ basic fibroblast growth factor (bFGF) (both PeproTech, Cranbury, NJ, USA) and 0.5 × B27 (Thermo Fisher Scientific)] to obtain a pure epithelial cell culture. Cells were then cultured adherently in DMEM supplemented with 10% FCS and antibiotic–antimycotic (Thermo Fisher Scientific) at 37 °C in 5% CO₂ in a humidified atmosphere.