Cells were incubated at 37 °C in humidified atmosphere (95% air, 5% CO2) in F-12K Nutrient Mixture (Kaighn’s Modification), 5% fetal bovine serum, and gentamicin (50 μg/ml) for 18–24 h. Cell staining was performed using the membrane permeable dye Calcein AM (Invitrogen, Karlsruhe, Germany), which, after uptake by the cell, shows green intracellular fluorescence. Only donor cells were

stained with 10 μM Calcein AM for 20 min at 37 °C, trypsinized, counted, and then added to a 96-well plate with (unstained) acceptor cells at a density of 4 × 103 cells/well. Lucifer Yellow could not be used for this method, because this dye does not enter an intact cell. Prior to the addition of donor cells, culture medium EGFR inhibitor was aspirated out. Solvent control (0.5% dimethyl sulfoxide

[DMSO]), positive control (phorbol-12-myristate-13-acetate [TPA]), or TPM was applied together with the stained donor cells, followed LY294002 chemical structure by centrifugation (300 × g for 5 min) of the plates and subsequent incubation for 3 h at 37 °C and 5% CO2. 10–12 Cigarettes were smoked on a 20-port rotary Borgwaldt smoking machine (RM20 CSR, Borgwaldt KC, Hamburg, Germany) according to ISO specifications, i.e., 35 ml puff volume, 2 s puff duration, 1 puff per minute for each cigarette (ISO, 2000). Total particulate matter (TPM) was collected on a Cambridge filter and dissolved in DMSO to a final concentration of 25 mg/ml DMSO. TPM from the Reference Cigarette 2R4F (Chen and Moldoveanu, 2003) a standard reference cigarette containing both Bright and Burley tobacco (University of Kentucky), and two specially designed single-tobacco

experimental cigarettes, i.e., a Bright cigarette and Burley cigarette, were applied to the cells. Both Janus kinase (JAK) the Bright and the Burley tobacco were of US origin. The TPM exposure concentrations from each cigarette were 0.02, 0.04, 0.06, 0.08, 0.1, and 0.12 mg/ml. DMSO (0.5%) was used as the solvent control. TPA (Sigma–Aldrich; Taufkirchen, Germany) which elicits a dose–response (see Fig. 3) was used at a concentration of 1 ng/ml as a positive control of GJIC inhibition. TPM from the 2R4F, Bright, and Burley cigarettes is cytotoxic (Roemer et al., 2004 and Roemer et al., 2009); therefore, prior to assessment of GJIC inhibition (only during dose-range–finding experiments), assessments of viability were performed to exclude cytotoxicity as a source of decreased gap junction activity. Ten μl/well of propidium iodide stock (50 μg/ml, Invitrogen, Karlsruhe, Germany) was used to determine the number of dead cells in response to 3-h exposure to TPM. Following the 3-h incubation period, culture medium was aspirated and cells were incubated in 100 μl/well fluorescent dye (Hoechst 33342, 10 μg/ml) for 10 min.