4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes

The human pancreatic cancer cell lines MIA PaCa-2, AsPC-1, PANC-1 and BxPC-3 were purchased from American Type Culture Collection (CRL-1420TM, CRL-1682, CRL-1469 and CRL-1687, respectively, ATCC; Rockville, MD, USA) and banked at Centre Paul Strauss. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. PANC-1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, PAN Biotech GmbH) and 1% of a solution of penicillin (10000 IU/mL) and streptomycin (10 mg/mL) (PAN Biotech GmbH). MIA PaCa-2 was cultured in the same conditions with 1% of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1 mM, PAN Biotech GmbH), 1% of sodium pyruvate (10 mM, PAN Biotech GmbH) and 1% of non-essential amino acids (NEAA, PAN Biotech GmbH). AsPC-1 and BxPC-3 were cultured in Roswell Park Memorial Institute medium (RPMI; PAN Biotech GmbH) supplemented with 10% FBS, and 1% of a solution of penicillin (10000 IU/mL) and streptomycin (10 mg/mL). Subconfluent cell monolayers were trypsinized once a week using 0.5% trypsin containing 2% EDTA (PAN Biotech GmbH) and plated at passage ratios between 0.25:10 to 1:10, according to the cell line or used directly for study after enumeration determined with a Countess^® Cell Counter (Countess, Invitrogen, Carlsbad, CA, USA).

If not otherwise stated, all chemicals were purchased from NeoFroxx. To maintain cells outside of the incubation chamber, a cell storage buffer containing 2 mM CaCl₂, 5 mM KCl, 138 mM NaCl, 1 mM MgCl₂, 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Pan-Biotech, Aidenbach, Germany), 0.44 mM KH₂PO₄, 2.6 mM NaHCO₃, 0.34 mM NaH₂PO₄, 10 mM D-Glucose, 0.1% minimum essential medium (MEM) Vitamins (Pan-Biotech, Aidenbach, Germany), 0.2% essential amino acids (Pan-Biotech, Aidenbach, Germany), 100 µg/mL Penicillin (Pan-Biotech, Aidenbach, Germany) and 100 U/mL Streptomycin (Pan-Biotech, Aidenbach, Germany) was used. The pH was adjusted to 7.43 using 1 M NaOH. The cell storage buffer was sterilized using a 0.45 µm medium filter (Isolab, Eschau, Germany). For live-cell imaging experiments, a HEPES-buffered solution was used, consisting of 2 mM CaCl₂, 5 mM KCl, 138 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, 10 mM D-Glucose, and pH was adjusted to 7.43 using 1 M NaOH.

Nicotine of analytical grade (≥ 99%), ammonium acetate (> 99%), ammonia (25%), Hank’s Balanced Salt Solution (HBSS), hydrochloric acid, and sodium hydroxide were obtained from Merck KGaA (Darmstadt, Germany). 2’,7’-Dichlorofluorescin diacetate (DCFDA) was obtained from Thermo Fisher Scientific (Schwerte, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM, P04-03596) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from PAN Biotech (Aidenbach, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent) was obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Milli Q Integral Water Purification System (Merck KGaA, Darmstadt, Germany) was used to prepare ultra-pure water.