Annexin 5 apoptosis assay

Sodium phosphate buffer saline (PBS, 0.1 M, pH = 7.4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMEM-F12 medium, senescence cells histochemical staining kit, and trypsin were received from Aldrich (Poznań, Poland). Hydrochloric acid (HCl, 35–38%) and 2-propanol (98%) were received from Chempur (Piekary Śląskie, Poland). Physiological saline without Ca&Mg (PBS, PAA, Poland), Annexin-V apoptosis assay (BioLegend, San Diego, CA, USA), Annexin V Binding Buffer (BD Biosciences, San Jose, CA, USA), and propidium iodide solution (BD Biosciences, San Jose, CA, USA) were used as received. Normal human dermal fibroblasts (NHDF) were obtained from Lonza (Lonza; Celllab, Warsaw, Poland). Malignant melanoma cells (Me45) and a human metastatic melanoma variant of the WM164 cell line (451-Lu) were obtained from the collection of the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology (Gliwice, Poland). Spontaneously immortalized human epidermal keratinocytes (HaCaT) were purchased from CSL Cell Line Service GmbH (Eppelheim, Germany). Copolymers were synthesized and self-assembled to encapsulate active substance in aqueous solutions [23 (link),24 (link)] or conjugated with FA and LA [25 (link)] according to previously described procedures.

Sodium phosphate buffer saline (PBS, pH = 7.4), DMEM-F12 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and trypsin were received from Aldrich (Poznań, Poland). Propidium iodide solution (PI, BD Biosciences, San Jose, CA, USA), Annexin-V apoptosis assay (BioLegend, San Diego, CA, USA), physiological saline (PBS without Ca and Mg, PAN-Biotech Gmbh, Aidenbach, Germany), fetal bovine serum (FBS, EURx, Gdańsk, Poland) and Annexin-V binding buffer (BD Biosciences, San Jose, CA, USA) were used without prior preparation. Human bronchial epithelial cells (BEAS-2B) and adenocarcinomic human alveolar basal epithelial cells (A549) were purchased from ATCC (Cat# ATCC^®CRL-9609; Manassas, VA, USA). Graft copolymers containing TMAMA units with chloride counterions were synthesized by controlled atom transfer radical polymerization, whereas their conjugates with p-aminosalicylic (PAS) or clavulanic (CLV) anions were obtained by chloride anion exchange, according to previously described procedures [27 (link)].

Tricyclic 10H-3,6-diazaphenothiazine (DPT-1, with two pyridine rings) and pentacyclic 7-(3′-dimethylaminopropyl)diquinothiazine (DPT-2, with two quinoline rings) were obtained according to previously described methods [20 (link),21 (link)]. The 1 mM stock of both phenothiazines was prepared in 100 % DMSO (Sigma Aldrich, Poznań, Poland), and before addition to the cells, the appropriate solutions, in fresh, complete sterile DMEM-F12, were prepared (final solutions used for biological experiments were as follows: 100, 50, 25, 12.5; 6.25; 3.15; 1.56; 0.78 µM). DMEM-F12 medium, trypsin, sodium phosphate buffer saline (PBS, pH = 7.4), and doxorubicin were bought from Merck (Poznań, Poland). Annexin-V apoptosis assay was obtained from BioLegend (San Diego, CA, USA). Propidium iodide solution (PI) was obtained from BD Biosciences (San Jose, CA, USA). Fetal bovine serum (FBS, EURx, Gdańsk, Poland), physiological saline (PBS without Ca and Mg, PAN-Biotech Gmbh, Aidenbach, Germany), and Annexin-V binding buffer (BD Biosciences, San Jose, CA, USA) were used after dissolving sterile H₂O 10 times prior to usage [23 (link)].