Gibco dmem f 12

Human U2OS cells expressing a tetracycline-regulated ERα (U2OS-ERα) were prepared, characterized, and maintained as previously described (Tee et al. 2004 (link)). The cells were maintained in phenol red-free Gibco DMEM/F-12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% charcoal–dextran stripped fetal bovine serum (FBS, Gemini Bio Products, West Sacramento, CA, USA), 100 units/mL penicillin and streptomycin, 50 μg/mL Fungizone, and 2 mM of glutamine. To maintain stable transfected cells, 50 μg/mL hygromycin B and 500 μg/mL of zeocin (Invitrogen, Waltham, MA, USA) were included in culture media. MCF-7 breast cancer cells were maintained in phenol red-free DMEM/F-12 supplemented with 10% FBS, 100 units/mL penicillin and streptomycin, 50 μg/mL Fungizone, and 2 mM of glutamine. For experiments, the culture medium was replaced with 5% charcoal–dextran stripped FBS in phenol red-free DMEM/F12.

Immortalized human proximal tubule epithelial (HK-2) cells were cultured in Gibco DMEM/F-12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 10 ng/mL epidermal growth factor (MilliporeSigma, Burlington, MA, USA), 5 μg/mL transferrin (MilliporeSigma), 5 μg/mL insulin (MilliporeSigma), 0.05 μM hydrocortisone (MilliporeSigma), 50 U/mL penicillin (Thermo Fisher Scientific), and 50 μg/mL streptomycin (Thermo Fisher Scientific). Cells were maintained at 37 °C with 5% CO2. HK-2 cells were subcultured in six-well plates and then starved of serum overnight. For phosphorylated ERK and p38 immunoblots, cells were treated with DMEM/F-12 medium for control and 125 ng/mL rhFSTL1 (Novus Biologicals NBP2-23056) in DMEM/F-12 medium for either 5, 10, 30, 60, or 120 min. For PARP and CASP3, cells were treated with DMEM/F-12 medium for control, 125 ng/mL rhFSTL1 in DMEM/F-12 medium for 16 h, or 1 μm of Naloxone in DMEM/F12 for 8 h the subsequently treated with 125 ng/mL rhFSTL1 in DMEM/F-12 medium for 16 h. For COX2, cells were treated with DMEM/F-12 medium for control or 125 ng/mL rhFSTL1 in DMEM/F-12 medium for 18 h. For COL1A1, cells were treated with DMEM/F-12 medium for control or 125 ng/mL rhFSTL1 in DMEM/F-12 medium for 48 h.

As demonstrated in Figure 1—figure supplement 1A, mice were euthanized as per MGH IACUC protocols, and trachea were dissected, cleared of connective tissue, opened longitudinally, and bisected. The dissected trachea was then sutured onto a silicone O-ring and placed onto a custom-made insert. The custom made insert was assembled using an inverted 12 mm Transwell, 0.4 µm Pore Polyester Membrane Insert fixed to a custom 3D printed insert (https://github.com/vss11/Label-free-autofluorescence) with Sylgard 184 silicone elastomer (EMS Catalog #24236–10).
Samples were incubated with Gibco DMEM/F12 (Fischer 11320033). For experiments across multiple days, the insert was filled with media such that the meniscus was located below the trachea to prevent explant submersion. Primocin (InVivoGen) was added to the media to prevent contamination.

Mouse pre-adipocytes within the stromal/vascular fraction (SVF) from subcutaneous iWAT were isolated from adult WT or transgenic AdipoR1 mice. Detailed procedures for pre-adipocyte isolation, cell culture and differentiation into mature adipocytes were described previously with slight modification [59 (link),60 (link),61 (link)]. Briefly, the mouse subcutaneous inguinal region of WAT was surgically dissected, minced and digested with collagenase (C6885, Sigma-Aldrich, St. Louis, MO, USA) and then filtered through chiffon. Pre-adipocytes within SVF were derived through a series of centrifugation and washing steps with red-blood-cell-lysis before the final washing step. Then, pre-adipocytes were suspended and seeded on tissue-culture treated plates containing medium of Dulbecco’s Modified Eagle Medium/F12 (Gibco DMEM/F12, Thermo Scientific), 10% fetal bovine serum (Thermo Scientific) and 1% antibiotics (penicillin, streptomycin and amphotericin B; Biological Industries, Kibbutz Beit-Haemek, Israel). Cells were incubated at 37 °C in air containing 5% CO₂ until confluency. Confluent pre-adipocytes were subsequently switched to induction medium with or without the PPARγ-agonist rosiglitazone for adipocyte differentiation. After around 7 to 9 d, mature adipocytes with at least 70% differentiation were collected for further analysis.

Mice used to source salivary glands were embryonic day 16 (E16) timed-pregnant CD-1 female mice from Charles River Laboratories (Wilmington, MA, USA). The care and handling of mice were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University at Albany, State University of New York. Primary E16 mesenchyme cells were isolated from mouse SMGs, as previously described [9 (link),18 (link),38 (link),39 (link)] and then grown in culture medium composed of Gibco DMEM/F-12 (Thermo Fisher Scientific, Grand Island, NY, USA), without HEPES or phenol red, supplemented with 10% FBS, 1% pen/strep (Sigma Aldrich). Cells were cultured in a 37 °C, 5% CO₂ humidified incubator and subcultured every 2–3 days for no more than 2 or 3 passages. The medium was typically replaced every other day.