35 mm petri dishes

CFBE-ΔF508 and CFBE-wt cell lines [CFBE41o- parental cells (Kunzelmann et al., 1993 (link)) stably transduced, respectively, with F508del-Cftr and wt-Cftr Bebok et al., 2005 (link)] were grown in EMEM medium (Wisent Inc., St-Bruno, QC, CA) supplemented with 10% FBS (Life technologies, Burlington, QC, CA), 2 mM L-glutamine (Life technologies) and 100U/ml of penicillin-streptomycin (Life technologies) on 35 mm petri dishes (Corning Inc., Corning, NY, USA), Lab-Tek 8 chamber slides (Thermo-Fisher Scientific Inc., Waltham, MA, USA) and permeant filters (4.67 cm²; Corning Inc.) coated with a LHC basal medium (Life technologies) solution containing 1 mg/ml BSA (Life technologies), 0.05 mg/ml bovine collagen I (Life technologies) and 1 mg/ml human fibronectin (VWR, Mont-Royal, QC, CA). Cells were cultured for 5 days on Lab-Tek chamber slides for immunofluorescence assays and 8 days on petri dishes before protein extraction, while cells on permeant filters were cultured for 3 weeks before electrophysiological measurements (Trinh et al., 2012 (link), 2015 (link); Bilodeau et al., 2016 (link)).

For osteogenic differentiation, 0.8 × 10⁵ cells suspended in lg-DMEM (ThermoFisher-Gibco) supplemented with 10% FBS were seeded in 35 mm Petri dishes (Corning). When 60% confluence was reached, induction was initiated with StemPro osteogenic medium (Gibco, Carlsbad, California, CA, USA), and the cells were incubated for 21 days, changing the medium twice per week. Finally, alkaline phosphatase activity was detected using SIGMA FAST^™ BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) (Sigma-Aldrich).

For microscopy, C6 glioma cells were plated in 35 mm Petri dishes (Corning Inc., Corning, NY, USA), 0.1 million cells in 2 mL of medium. Nicotine was added at final concentrations of 0.001, 0.01, 0.1, 1 and 10 μL/mL, in the control–solvent (DMEM medium with 10% FBS). After 24, 48 and 72 h, microscopy was performed using an NU2E inverted phase contrast microscope (Opton, Oberkochen, Germany) with a Nicon LWD 40 × 0.55 objective (Nikon Corporation, Tokyo, Japan). For photography, an XLI-14A camera from XL Imaging (XL Imaging LLC, Carrollton, TX, USA) with the supplied software was used.

HeLa (human cervical cancer cells) and A549 (human lung carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 2.5 mM L-Glutamine (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 IU/mL penicillin (Sigma Aldrich, Darmstadt, Germany), and 100 μg/mL streptomycin (Sigma Aldrich, Darmstadt, Germany) at 37 °C in 5% CO₂.
Prior to irradiation, the cells were seeded at a density of 0.4 × 10⁵ cells/mL in 2.5 mL of culture medium onto coverslips (Thermo Fisher Scientific, Waltham, MA, USA) placed inside 35 mm Petri dishes (Corning, New York, NY, USA) and incubated at 37 °C and 5% CO₂ for 20 h.

HEK 293 cells stably expressing human α4β2 nAChRs were cultured by standard methods (Di Resta et al., 2010 (link)). In brief, cultures were maintained in Dulbecco's modified Eagle's medium (Hyclone Laboratories), supplemented with 10% fetal calf serum (Hyclone), 4.5 g/l glutamine, 0.05 ng/ml hygromycin β, and 0.25 μg/ml amphoterycin B. Cells were grown at 37°C and 5% CO₂. For patch-clamp experiments, cells were harvested by treatment with trypsin and plated onto 35 mm Petri dishes (Corning Inc.).

On day –2, 0.8×10⁶ mature BMDMs were seeded on non-tissue cultured treated 35 mm petri dishes (Corning) and activated as previously outlined on Day –1. The naive or activated/trained BMDMs were harvested at two separate time points: day 0 (24 hours post activation for acute activation characterisation) or day 6 (fed on day 3), with or without secondary stimulation on day 5 as specified in figure legends (training experiments).
For analysis, BMDMs were placed on ice for 30 min before harvesting with PBS-EDTA (5 mM) solution by gently pipetting up and down and transferring to flow cytometry tubes. Cells were incubated with Fixable Viability Stain 510 at RT for 15 min. After washing with PBS, cells were stained with anti-mouse Fc block, 15 min prior staining with CD80-FITC, CD206-PE, F4/80-PerCP-Cy5.5, CD11b-APC-eFluor 780, MHC class II-eFluor 450 for an additional 30 min at 4 °C. The cells were washed with PBS and resuspended in flow cytometry buffer (1% FBS in PBS). Samples were acquired on a BD Canto II flow cytometer and the data was analysed by using FlowJo software.