Ethylene diamine tetraacetic acid (edta)

Mouse macrophage primary cell cultures were prepared as described previously [23 (link)]. Procedures with BALB/c mice were performed at Vilnius University Life Science Center animal facility (Vilnius, Lithuania) in accordance with EU legislation (State Food and Veterinary Service permission No. LT 61-13-004). Spleens were separated from the abdominal cavity of 6- to 8-week-old female BALB/c mice, crushed, and filtered through a 100 μm nylon strainer. Lysis buffer was used to remove red blood cells and cell suspension was prepared. Then, splenic cells were analysed by flow cytometry to determine the count of CD11b⁺ cells. CD11b⁺ cells were cultured at a density of 0.3 × 10⁶ per well for 5 days in RPMI 1640 + 10% FBS + 2 mM L-glutamine + 100 μg/ml gentamicin and 10 ng/ml M-CSF (Invitrogen, USA) in 12-well plates, at 37°C with 5% of CO₂. For macrophage activation on day 5^th, cells were stimulated with LPS (50 ng/ml) and IFN-γ (20 ng/ml) or IL-4 (20 ng/ml) and IL-13 (20 ng/ml) to obtain either M1 or M2 phenotype, respectively. After 48 h of incubation, cell culture supernatants were collected and stored at −20°C for further cytokine analysis. The cells were harvested by trypsin 0.05% and 0.02% EDTA (Biochrom, Germany) for flow cytometry analysis.

MCF10 human breast cell lines, MCF10A and MCF10CA, were cultured in DMEM/F12 medium (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 5% horse serum (Invitrogen, Carlsbad, CA, USA), 2 mM glutamine (EuroClone, Pavia, Italy), 100 µg/mL streptomycin sulfate, 100 U/mL penicillin (EuroClone), 0.5 µg/mL epidermal growth factor (EGF, Sigma), 0.5 µg/mL hydrocortisone (Sigma), 0.1 µg/mL cholera toxin (Sigma), and 10 µg/mL insulin (Sigma). Both cell lines were passaged twice a week at the following split ratios (MCF10CA 1:20; MCF10A 1:25) using 0.05% trypsin (w/v) 0.02% EDTA (w/v) (Biochrom AG, Berlin,Germany). For experiments with the EV isolation, serum was purified by centrifugation at 110,000× g for 16 h and sterile-filtered with 0.22 µm syringe filters (Guangzhou Jet Bio-Filtration Co., Ltd., Guangdong, China).

Human skin biopsies were incubated with 2.2 U/mL dispase II (Roche) overnight at 4 °C. After removal of the epidermal layer, the dermis was digested with 2.5 mg/mL collagenase (Sigma, Darmstadt, Germany) for 45 min at 37 °C. Cell suspension was passed through a 70 µm filter. Cells were cultured at 37 °C, 5% CO₂ in DMEM medium (Anprotec, Bruckberg, Germany) containing 10% fetal calf serum (FCS; PAN-Biotech, Aidenbach, Germany) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany). After reaching confluence, cells were passaged using 0.05% trypsin and 0.02% EDTA (Biochrom). Cells were stimulated with 10 ng/mL human IL-4 (Miltenyi, Bergisch-Gladbach, Germany), IL-6 (Miltenyi), IL-13 (Miltenyi), or TNFα/IL1β (Miltenyi) or 20 ng/mL TGFβ1 (Peprotech, Hamburg, Germany) in DMEM/0.5 % FCS for 9d. Medium was changed after 7d. To exclude any effects of proliferation or cell death, the cell number was counted, and the sThy-1 concentration was presented as pg per 10,000 cells.

Human PDAC cell lines BxPC3, Panc-1, PancTu-I and Capan-2 derived from primary tumors (stage G1–G3), Panc89 and Colo357 cells established from lymph node metastases (stage G1–G2) as well as Capan-1 cells generated from liver metastases (stage G1) were cultured in complete medium under regular conditions (5% CO₂, humidified, 37 °C). The PDAC cell lines were kindly provided by Dr. C. Röder, Prof. Dr. A. Trauzold and Prof. Dr. S. Sebens, Institute for Experimental Cancer Research, Kiel, Germany. Human breast cancer cell line MCF-7 (ATCC, Manassas, VA, USA) was also cultured in complete medium. For detachment of adherent tumor cells, 0.05% trypsin/0.02% EDTA (Biochrom) was used. Genotyping of tumor cells was carried out by short tandem repeat analysis and absence of mycoplasma was routinely confirmed by RT-PCR (Venor^® GEM classic, Minerva Biolabs GmbH, Germany).

The human breast adenocarcinoma cell line, MCF-7, was cultured in minimum essential medium alpha (MEMα, EuroClone, Pavia, Italy) supplemented with 5% fetal bovine serum (FBS, HyClone, Thermo Scientific, Epsom, UK), 2 mM glutamine (EuroClone), 50 μg/mL streptomycin sulfate, and 50 U/mL penicillin (EuroClone). Cells were passaged twice a week at a 1 : 5 split ratio using 0.05% trypsin (w/v) 0.02% EDTA (w/v) (Biochrom AG, Berlin, Germany).

The cells irradiated with protons and X-rays were processed at the same time. About 1 h after irradiations, the cells were detached with trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05 %/0.02 %, Biochrom, Berlin, Germany) and counted. Depending on the irradiation dose and the expected SF, an appropriate number of cells (500–50,000) was plated onto 100-mm Petri dishes (Falcon; Franklin Lakes, USA, three dishes per dose) in 15 ml of medium, to give 50–100 colonies per dish. After incubation at 37 °C for 18 days, the resultant colonies were washed with 0.9 % sodium chloride (NaCl), fixed in 80 % ethanol (both Chempur, Piekary Śląskie, Poland) and stained with 1 % crystal violet (Merck, Darmstadt, Germany). Colonies consisting of more than 50 cells were scored as representing surviving cells. The SF at each dose point was calculated as an average of three plates. The plating efficiency (PE) of non-irradiated HFIB2 cells was ~25 %, while that of HFIB15 and HFIB30 was ~30 and ~21 %, respectively. SF was calculated as the ratio of PE for irradiated and non-irradiated cells.

ACL samples (one female donor, age 40), were harvested in accordance with the institutional ethical committee of the Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin (ethical approval number EA4-033-08, approval date: 22 May 2008). Surrounding connective tissue, including the synovial membranes, was removed. The pure ligament tissue was cut into 1–2 mm² slices and incubated in T25 cell culture flasks (Sarstedt, Nümbrecht, Germany) with growth medium (Ham’s F-12/Dulbecco’s Modified Eagle’s [DMEM] Medium 1:1) containing 10% fetal calf serum (FCS), 10,000 IU/mL penicillin/10,000 µg/mL streptomycin, 2.5 µg/mL amphotericin B, non-essential amino acids (all from Biochrom, Berlin, Germany) and 25 µg/mL ascorbic acid (Sigma-Aldrich, Munich, Germany) at 37 °C and 5% CO₂. After 1–2 weeks, the ACL ligamentocytes started to migrate from the tissue slices. Subsequently, ligamentocytes were harvested using 0.05% trypsin/0.02% EDTA (Biochrom) and expanded in T75 and T175 culture flasks. For analyzing the expression of marker protein profile on protein level, ACL ligamentocytes were seeded on poly-L-lysin (Biochrom) coated cover slips at 1 × 10⁴ cells/cm².