Fetal bovine serum (fbs)

Cell culture HT-29 (HTB-38), DLD-1 (CCL-221) human colorectal adenocarcinoma cell lines and fibroblasts skin cells were acquired from the American Type Culture Collection (ATCC). First cell line was grown in McCoy’s 5A medium (Pan Biotech, Aidenbach, Lower Bavaria, Germany), the second cell line was maintained in RPMI 1640 medium (ATCC, Manassas, VA, USA), and the third one in DMEM (Corning, Kennebunk, ME, USA). The growth supplement for cell culture (fetal bovine serum (FBS—Eurx, Gdansk, Poland)) and antimicrobial substances (penicillin/streptomycin)(Corning, Kennebunk, ME, USA) were added in 10% and 1% concentration, respectively. The incubator asserted appropriate growth conditions which are required for this cell lines: 5% of carbon dioxide, 37 Celsius degree and the humidity between 90% and 95%. The 100 mm plates were used to culture the cells. After a cell line reached about 80–90% confluency, the detachment of cells with 0.05% trypsin containing 0.02% EDTA (Corning, Kennebunk, ME, USA) and PBS (Corning, Kennebunk, ME, USA) was performed. The cells were then reseeded at density 5 × 10⁵ cells per well in six well plate in 1 mL of appropriate medium and after 24 h incubation used in the presented tests.

HCT116 cells (human colorectal cancer, ATCC-CCL-247) and Me45 cells (human melanoma, Oncology Center, Gliwice described in [53 (link)]) were grown in standard conditions in DMEM/F12, (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Eurx, Gdansk, Poland) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere with 5% CO₂, passaging and medium change were conducted every two days.

NCI-H1299 (non-small cell lung carcinoma, CRL-5803), MCF7 (breast carcinoma, HTB-22), and FaDu cells (squamous cell carcinoma, pharynx, HTB-43) were purchased from ATCC (Manassas, VA, USA). HaCaT (spontaneously immortalized keratinocytes) cell line was acquired from CSL Cell Line Service GmbH (Eppelheim, Germany). Cells were cultured at 37 °C under standard conditions (5% CO₂, 95% humidity, 21% O₂ concentration). NCI-H1299, MCF7 and FaDu cell lines were grown in RPMI, DMEM-F12 or MEM (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) respectively. HaCaT cells were grown in DMEM (high glucose 4.5 g/L; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Cell culture media were supplemented with 10% heat-inactivated fetal bovine serum (EuRx, Gdańsk, Poland) and antibiotics (gentamycin or penicillin-streptomycin). Cells were regularly checked for mycoplasma contamination.

NHDF-Neo, HCT 116, K562, Raji, BL (Ca46 and ST486) cells were obtained from ATCC. The Me45 cell line was derived in 1997 from a lymph node metastasis of skin melanoma of a 35 year-old male at the Radiobiology Department of the Centre of Oncology in Gliwice [7 (link)]. Cells were grown at 37°C in a humidified atmosphere with 5% CO₂ using DMEM/F-12 medium (GE Medical Systems Polska) for HCT 116, Me45 and NHDF-Neo cells and RPMI (Sigma-Aldrich) for K562, Raji, Ca46 and ST486 cells, supplemented with 10% fetal bovine serum (Eurx) and penicillin-streptomycin (Sigma-Aldrich). Cell doubling times were determined by counting cells in a hemocytometer. Cells were harvested in the exponential phase of growth for DNA isolation.

Blood samples were collected in two different seasons when the concentration of PM 10 in the air of Krakow was lower than the daily limit of 50 µg/m³ (summer), and when it was higher than 50 µg/m³ (winter). Peripheral blood mononuclear cells (PBMCs) were isolated by standard Pancoll (Panbiotech, Aidenbach, Germany) density gradient centrifugation, washed and resuspended in RPMI 1640 medium (Corning, Manassas, VA, USA), supplemented with 2 mM of L-glutamine, 5% heat-inactivated fetal bovine serum (EURx, Gdańsk, Poland), and 25 µg/mL gentamycin (Sigma, St. Louis, MO, USA) (complete medium).

BEAS-2B (virus-transformed non-tumorigenic bronchial epithelial cells; CRL-9609) and cancer cell lines: NCI-H1299 (non-small cell lung carcinoma, CRL-5803), NCI-H23 (adenocarcinoma, CRL-5800), NCI-H358 (bronchioalveolar carcinoma, CRL-5807), MCF7 (breast carcinoma, HTB-22), and HeLa (cervical carcinoma, CCL-2) were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37 °C under standard conditions (5% CO₂, 95% humidity, 21% O₂ concentration). BEAS-2B cells were grown in serum-free bronchial epithelial growth medium (Lonza Ltd, Basel, Switzerland) and maintained at low density (less than 70% confluence). NSCLC cell lines were cultured in RPMI (Sigma-Aldrich, St. Louis, MO, USA), MCF7 in DMEM/F12 media (Sigma-Aldrich, St. Louis, MO, USA), and HeLa in RPMI/DMEM-HG (1:1 v/v), all growth media were supplemented with 10% heat-inactivated fetal bovine serum (EuRx, Gdańsk, Poland) and antibiotics (gentamycin or penicillin–streptomycin, all experiments were performed without antibiotics addition). Cells were regularly checked for mycoplasma contamination.

Two breast cancer cell lines (MCF-7 and MDA-MB-231) and fibroblasts as normal cell lines, which were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were used to investigate the effects of the novel synthesized compounds. The cells were cultured in DMEM (Corning, Kennebunk, ME, USA), supplemented with FetalBovine Serum (Eurx, Gdansk, Poland) at concentration 10% and penicillin/streptomycin at 1%. Cells were cultured on 100 mm plates (Sarstedt, Newton, NC, USA) and then kept in an incubator that provided optimal growth conditions (5% CO₂, temperature: 37 °C, humidity at 90–95% level). After reaching 80% confluence, cells were detached from the bottom of the plate using 0.05% trypsin supplemented with 0.2% EDTA (Gibco, San Diego, CA, USA). Then, using a hemocytometer, the number of cells was quantified and seeded at density 5 × 105 cells per well in six-well plates (Sarstedt, Newton, NC, USA) in 2 mL of DMEM. In this research, cells that obtained 80% of confluency were used.