Streptomycin antibiotic

The NIH3T3 cell line and Lck infectants cell lines were maintained in Iscove’s modified Dulbecco’s medium (IMDM), WT, and JCAM1.6 The Jurkat T-cell line and RAJI B-cell line were cultured in RPMI 1640 (Life Technologies). Both media were supplemented with 10% inactivated fetal calf serum (FCS) and 100 U of penicillin/10 μg streptomycin antibiotics (Sigma) per 1 ml of media. Bone marrow-derived dendritic cells (BMDCs) from OTII transgenic mice were isolated from mice femur and tibia cavities. The cells were cultured for 6 days in RPMI medium supplemented with GM-CSF-containing supernatant, which was produced by the LUTZ cell line (final concentration was adjusted to 30 ng/ml). After 3 days of cultivation, one half of the media was replenished, and on day 6, the cells were harvested and used for further experiments.

The human mesenchymal amniotic fluid stem cells (AFSC) were used to evaluate the biocompatibility of ZnO nanoparticles. The cells were cultured in DMEM medium (Sigma–Aldrich, Missouri, MO, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma–Aldrich, Missouri, MO, USA). To maintain optimal culture conditions, the medium was changed twice a week. The biocompatibility was assessed using MTT assay (Vybrant^® MTT Cell Proliferation Assay Kit, Thermo Fischer Scientific, Massachusetts, MA, USA). The assay is a colorimetric method that allows quantitative assessment of proliferation, cell viability and cytotoxicity. The viable cells reduce yellow tetrazolium salt MTT (3- (4,5dimetiltiazoliu) -2,5-diphenyltetrazolium bromide) to a dark blue formazan via mitochondrial enzymes. Briefly, the AFSC were grown in 96-well plates, with a seeding density of 3000 cells / well in the presence of the analyzed samples (5 mg/mL concentration of each ZnO nanopowder) for 72 h. Then, 15 mL of Solution I was added and incubated at 37 °C for 4 h. Solution II was added and pipettes vigorously to solubilize formazan crystals. After 1 h, the absorbance was read using a spectrophotometer at 570 nm (TECAN Infinite M200, Männedorf, Switzerland).

The cell culture medium consisted of Dulbecco‘s modified Eagle‘s medium (cat. No. D5546, Sigma-Aldrich Chemie, Steinheim, Germany), 9% fetal bovine serum—FBS (F7524, Sigma-Aldrich Chemie), 1% L–glutamine solution (G7513, Sigma-Aldrich Chemie), 100 U/mL penicillin, and 90 μg/mL streptomycin antibiotics (P0781, Sigma-Aldrich Chemie). The cell electroporation medium was Minimum Essential Medium Eagle, Spinner Modification (S-MEM) (M8167, Sigma-Aldrich Chemie). Altogether, 0.9% NaCl (Balkanpharma-Troyan AD, Troyan, Bulgaria), FeCl₂, and FeCl₃ (Fluka Chemie GmbH, Buchs, Switzerland) were used to prepare the solutions of 0.9% NaCl with various concentrations of Fe²⁺ and Fe³⁺.