Ciproxin 200

For the preparation of the autologous vaccine, melanoma cell lines were established from resected metastases. All patients gave their informed consent to receive the vaccine. The melanoma cell lines were established and cultured as described [18 (link)]. Briefly, cells were extracted mechanically from fresh and sterile tumor specimens, frozen, and stored in liquid nitrogen in a medium containing 2.5% human albumin and 20% DMSO until needed. To assure melanocytic progeny, the expressions of S100, MART-1, and gp100 were determined by immunostaining using polyclonal rabbit anti-S100, monoclonal A-103, and HMB-45 Abs, respectively (Dako). Positive staining of more than 50% of cells with at least one of these antibodies was required. MHC class I-related chain A (MICA) expression was determined by flow cytometry of melanoma cell lines using anti-human MICA-APC (Allophycocyanin), R&D systems, Minneapolis, MN, USA.
Cell lines were routinely tested for mycoplasma contamination by EZ-PCR (Biological Laboratories, Beth Haemek, Israel), according to the manufacturer's instructions. Tumor cultures that were found contaminated were incubated in the presence of 10 mg/mL Ciproxin 200 (Bayer) for two weeks, with change of medium every three days. The cells were retested after treatment and were used only after being found mycoplasma-free.

Micro-Agar-PCR-test was performed using MHA (BD 225250) for B. anthracis and Y. pestis and CHA for F. tularensis.Agar media was prepared according to the manufacturer’s instructions. Agar dilution was performed essentially as described in CLSI standard M07 (CLSI, 2018 ). Following autoclaving, the agar was chilled to 50°C and 40 ml were aliquoted to 50 ml tubes where the tested antibiotic doxycycline (Sigma D9891) and ciprofloxacin (ciproxin 200, Bayer) was added. 10X of antimicrobial solution was diluted by making twofold serial dilutions in master tubes. Next, one part of the 10X antimicrobial solution was added to nine parts of molted agar. Agar with no antibiotics served as growth control. 150 μl aliquots of the antibiotic-supplemented melted agar were divided into a 96-well plate.

ASTs were performed using the standard microdilution method [35 ] using ciprofloxacin solution (Ciproxin 200, Bayer), ampicillin (Sigma A0166), gentamicin (Sigma G1264) and doxycycline (Sigma D9891). For susceptibility tests, stock solutions were serially diluted two-fold in cation-adjusted Müeller-Hinton broth (CAMHB, BBL 212322) and placed in 96-well flat bottom microtiter plates (TPP 92696). 50 μl of freshly prepared bacterial suspensions in CAMHB were inoculated in triplicates with 5*10⁵-1*10⁶ CFU/ml in a final volume of 0.1 ml containing antibiotic concentrations in the range of 0.03–16 × MIC. Cultures were incubated for 24 h at 28 °C in a plate reader (Sunrise or Infinite F-200 pro, TECAN), and the optical density at 630 nm (OD630) was read at 1-h intervals. The MIC values were defined after 24 h as the lowest antibiotic concentration that reduced growth to less than 10% of the OD at 630 nm of the growth control (without antibiotics). Lack of growth in MIC wells was verified visually by naked eye inspection. Each assay was performed in three independent experiments. For the time-lapse susceptibility analysis by FACS, similar AST conditions were used in a 24-well flat bottom microplate (Costar #3524) in a final assay volume of 0.5 ml.