Accutase

Control or acid-adapted cancer cells were plated at 150 cells/cm² on a low-attachment 100 mm plate in DMEM/F12 supplemented with N2, 5 g/ml insulin, 20 ng/ml FGF-2, and 20 ng/ml EGF (all from Thermo Fisher Scientific) at standard pH. Cells were allowed to form tumorspheres by 10–15 days. For serial passaging, 10-day-old tumorspheres were dissociated in single cells with Accutase (Euroclone), and plated at 150 cells/cm² on a low-attachment 100 mm plate for 10 additional days.

Primary skin fibroblast cell lines were isolated from skin biopsies as follows. Skin specimens were washed with 70% ethanol and physiological solution and incubated with 2 mg/mL Dispase II (Merck KGaA, Darmstadt, Germany) overnight at 4 °C. Then, the dermis was separated using sterile tweezers, cut into 2–4 mm pieces and plated on a 6-well microplate. A squared sterile glass was put above and 1 mL of high glucose Dulbecco’s modified Eagle’s medium, DMEM (Euroclone, Milan, Italy) supplemented with 20% fetal bovine serum, FBS (Euroclone, Milan, Italy) was added to each well. After 3 weeks, dermis and glasses were removed and fibroblasts were detached with 0.25% Trypsin and 0.02% EDTA (Euroclone, Milan, Italy) and plated in 25 cm² flasks. Cells were then cultured in DMEM supplemented with 15% (v/v) FBS, 100 U/mL penicillin, 100 mg/mL streptomycin (Euroclone, Milan, Italy) and 2 mM L-glutamine (Euroclone, Milan, Italy) and maintained at 37 °C under humidified conditions and 5% CO₂. Cells were sub-cultured twice weekly, detached with Accutase (Euroclone, Milan, Italy) and centrifuged at 500× g for 10 min at room temperature. Cells were used for proteomics analysis at passage number lower than 10. Cell pellets were collected, washed in PBS, frozen in liquid nitrogen and stored at −80 °C.

Cells were harvested by using Accutase (Euroclone, Milan, Italy), collected in flow cytometer tubes (2 × 10⁵ cells/tube), and stained 1 h at 4 °C with anti-CD133 (Affimetrix eBioscience, part of Thermo Fisher Scientific, Monza, Italy), anti-CD243 (Affimetrix eBioscience), anti-CD34 (BD PharMingen, San Diego, CA), anti-CD105 (Ancell, Bayport, MN), anti-CD73 (BD PharMingen), and anti-CD90 (BD PharMingen) antibodies. For intracellular antigen detection, cells were permeabilized for 15 min with 0.25% Tryton X-100 PBS, and then incubated 1 h at 4 °C with anti-ALDH1A1 (Abcam, Milan, Italy), anti-Nanog (GeneTex, CA, USA), anti-KLF4 (GeneTex), anti-OCT4 (GeneTex), and anti-SOX2 (GeneTex) antibodies. Cells were washed in PBS and incubated 1 h in the dark at 4 °C with secondary antibodies conjugated with FITC (Merk Millipore, Milan, Italy), Alexa Fluor 488 (Thermo Fisher Scientific, Monza Italy), PE (Immunotools, Germany), or APC (Immunotools). Samples were washed in PBS and analyzed at BD FACSCanto (BD Biosciences, Milan, Italy). The flow cytometer was calibrated using cells incubated with secondary antibody only. For each sample, 1 × 10⁴ events were analyzed.

Bulk multifluorescent cell lines were single cell-flow sorted using a BD FacsAria^TM III instrument (BD Bioscience) where single cells were sorted unbiased by any marker expression directly into the inner 60 wells of 5 laminin (Millipore) pre-coated flat-bottom 96-well plates (PerkinElmer). Single cells were dropped in 100 μL/well of the same medium as described above. The outer 16 wells were filled in with 200 μL/well of PBS to avoid evaporation of medium. After single cell-flow sorting, the plates were incubated at 37 °C, 5% CO₂, and colonies monitored as previously described [12 (link)]. Once weekly cells were refed with 25 μL of medium/well and plates scanned on a CeligoS cytometer (Nexcelom Bioscience, Lawrence, MA, USA) using the Confluence application, to evaluate colony growth. Single cell-derived colonies were detached using Accutase (Euroclone, Pero, Italy) when they achieved approximately 60–80% confluency and collected for further expansion to stably derive single cell-derived clones.

Internalization of nintedanib was determined by exploiting the fluorescent emission of nintedanib by means of a cytofluorimetric assay [35 (link)]. Briefly, A549 cells were seeded in standard medium (2 × 10⁶ cells/100 mm Petri dishes, Sarstedt), and after adhesion, cells were grown in the presence or in the absence of TGFβ (10 ng/mL) for 48 h. Next cells were exposed to nintedanib or conjugated compounds at different concentrations. After 24 h, cells were gently detached using Accutase (ECB3056D, Euroclone, Pero, Milano, Italy) or trypsin and the fluorescence, associated with different cell populations, was evaluated using the BV480 channel of the FACSCanto BD instrument (using 405 nm laser). Untreated cells were used as the negative control. The same procedure was applied to evaluate the internalization in K562, SSM2c, and L929 cells.

Flow cytometry analyses were performed in A375 adherent cells and in melanosphere-derived cells, in order to assess the expression of CD271 and ABCG2, and the autofluorescence level of the cells. Melanospheres were disaggregated by accutase (Euroclone) incubation. Single cells were resuspended in ice cold PBS containing 2% FBS (5 × 10⁵ cells and 2.5 × 10⁵ cells, respectively), and incubated with PE-conjugated anti-CD271 antibody or/and Alexa Fluor 405-conjugated anti-ABCG2 antibody. Cells were then washed with ice cold PBS and analyzed with excitation wavelengths of 488 (CD271) and 405 (ABCG2) nm, and a collection filter of 585/40 and 445/45 nm, respectively.
The autofluorescence analysis was performed without labeling the cells, using an excitation wavelength of 488 nm and the FITC collection filter (530/30), as previously described^{17 (link)}. The flow cytometry analysis was performed with a Novocyte3000 instrument (ACEA Biosciences, San Diego, CA). Data were analyzed with Novoexpress software.

Primary skin fibroblast cell lines were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Euroclone) supplemented with 15% (v/v) fetal bovine serum (FBS) (Euroclone), 100 U/ml penicillin, 100 μg/ml streptomycin (Euroclone), and 2 mM L-glutamine (Euroclone) and maintained at 37°C under humidified conditions and 5% CO2. Cells were subcultured twice weekly, detached with Accutase (Euroclone), and centrifuged at 500 × g for 10 min at 25°C. Cells were used at passage number lower than 13.
Fibroblast cells were seeded at a density of 5 × 10⁵ per 75 cm² flask for 24 h before treatments. Cells were then exposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissolved in dimethyl sulfoxide (DMSO) at a concentration of 60 μM or to an equal volume of DMSO alone, for 24 h.