Trypsin edta

The experiments were performed on normal and cancer cell lines, purchased from ATCC^®, Washington, DC, USA (U87MG, EOC2).
U87MG cells were cultured in DMEM (Sigma-Aldrich, Weinheim, Germany), supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). EOC2 cells were cultured in DMEM, supplemented with 10% FBS. Cells were cultured in a humidified atmosphere at 37 °C, saturated with 5% CO₂.
To remove the adhesive cells from the plates, we used a trypsin/EDTA solution (0.05% of trypsin/EDTA; Sigma-Aldrich, Weinheim, Germany) for U87MG, and a cell scraper for EOC2.
The cells were collected by centrifugation (125× g for 10 min) and placed in a fresh medium without antibiotics prior to treatment with the respective substance. The cells (3 × 10⁵ cells/mL) were incubated with the drug for different time intervals in a cell incubator. At each time interval, aliquots were used for analyses.
Ascorbate was dissolved in PBS (10 mM, pH 7.4). Menadione was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Weinheim, Germany) to 10 mM stock solutions, and then several working solutions in PBS were prepared. The final concentration of DMSO in the cell suspension was below 1%. At this concentration, DMSO did not affect cell viability.

Healthy cell line PNT2 was purchased from Sigma-Aldrich, while the metastatic prostate cancer cell line DU145 was obtained from ATCC-LGC Standards. Both suppliers ensure authenticated, validated and mycoplasma free cell lines. Cells were cultured in a lab with no history of mycoplasma infection after no longer than 3 months of purchase. DU145 cells were cultured in Corning T-75 flasks (VWR) in Dulbecco’s Modified Eagle Medium/DMEM containing 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (all Sigma-Aldrich)⁴³ . PNT2 cells were cultured in Corning T-75 flasks using RPMI-1640 medium (Sigma-Aldrich)⁴³ . Flasks were kept in a CO₂ controlled incubator at 37 °C. Samples were prepared for analysis once 70% confluent by removing supernatant media, washing each flask with 5 mL Trypsin-EDTA (Sigma-Aldrich) and incubating with 3 mL Trypsin-EDTA until cells had detached. Trypsin was neutralized adding 7 mL pre-warmed complete RPMI or DMEM⁴³ . After detachment, cells were spun for five minutes at 1500 RPM and the resulting pellet gently resuspended in 2 mL pre-warmed complete DMEM or RPM. Cells were passaged every third day.

Porcine adipose tissue-derived stromal cells (pADSCs) were isolated, characterized, and expanded in DMEM medium enriched by 10% (v/v) fetal bovine serum (FBS, Sigma, Munich, Germany) and antibiotics following published protocols [32 (link)]. In brief, the tissue was minced and incubated with 0.1% of collagenase (Gibco) and 1% of bovine serum albumin (BSA, 1% (w/v) in PBS) at 37° for 30 min. Incubation was stopped with medium containing 10% of FBS. The adipocyte phase was discarded and the stromal cell fraction was filtered through a 100 µm cell strainer. Cells were washed again with medium and the retrieved cells were seeded and incubated in expansion media containing 10% of FBS and antibiotics [32 (link)]. When reaching 70% of confluence, cells were detached (Trypsin-EDTA, Sigma, Munich, Germany), washed twice with PBS, and seeded in 10 mL of medium at an inoculation density of 3E05 ADSCs per 75 cm² flask. Cell proliferation and duplication rates (DR) were determined by cell counting over three consecutive passages [33 (link)]. To determine the mean size of pADSCs in suspension, cells were detached by Trypsin-EDTA (Sigma, Munich, Germany) and washed with phosphate-buffered saline (PBS, Sigma, Munich, Germany). Cell viability and dimensions were determined with a cell analyzer following the manufacturer’s instruction (CASY, Omni Life Science, Bremen, Germany).

Animals expressing GFP within the kidney were identified by fluorescent microscopy. The pronephric primordium was identified by anatomical position and explanted using sharp forceps from stage 30–32 embryos under white light. All explants were carried out on 2% agar-coated plates. Approximately 10–20 kidney explants were disassociated in Calcium Magnesium Free Media (CMFM) [1/3× MMR without MgSO₄ and CaCl] for 1 h. Once dissociated cells were washed in Danilchik’s for Amy (DFA) [53 mM NaCl, 5 mM Na₂CO₃, 4.5 mM potassium gluconate, 32 mM sodium gluconate, 1 mM CaCl₂, 1 mM MgSO₄, 0.1% (w/v) BSA, buffered to pH 8.3 with 1 M bicine] and allowed to attach. Dissociation and attachment procedures were carried out in Attofluor™ Cell Chamber (Thermo Fisher A7816, Waltham, MA, USA) with fibronectin-coated coverslips (Roche 10838039001, Basel, Switzerland). Alternatively, to increase the yields of individualized cells, explants were treated with Trypsin-EDTA (Ethylenediaminetetraacetic acid) (Sigma T4049, St. Louis, MO, USA) for 5 min instead of CMFM, followed by inactivation of Trypsin-EDTA with 10% fetal calf serum in DFA.

A single cell suspension was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich, #T3924), and manual disaggregation (25 gauge needle) to create a single cell suspension [5 (link)]. Cells were plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12/B27/20ng/ml EGF/PenStrep) in non-adherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma, #P3932). Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres >50 μm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation, and was normalized to one (1 = 100 %MSF). For proteomic analysis, mammospheres were collected by centrifugation at 800 rpm for 10 minutes.