Millex gv filter

The BPs were isolated by using the traditional enrichment method [29 (link)]. The water samples (2 mL, collected from the Ganges River) were centrifuged at 12,000× g for 10 min to remove debris and coarse material. The supernatant was then serially passed through membrane filters with pore sizes of 0.45 μm and 0.22 μm (Millex-GV Filter, Merck, Darmstadt, Germany) [30 (link)]. The processed water samples were then dropped on lawned A. baumannii bacterial Mueller–Hinton agar (MHA) petri dishes and incubated overnight at 37 °C. After incubation, the petri dishes were examined for the presence of lytic bacteriophage plaques and harvested with TMG buffer. The culturing and harvesting procedures were repeated until a total bacterial clearance and a higher BP (BPABΦ1) titer were obtained. The BPs were then purified by a single sequential plaque isolation method using a double-layer agar overlay technique (DLAO) [31 (link)].

In our experiments, we used colorectal adenocarcinoma HT29 (ATCC HTB-38), cervical adenocarcinoma cells HeLa (ATCC CRM-CCL2), and osteosarcoma MG63 (ATCC CRL-1427). The cells were maintained in Dulbeco’s Modified Medium (DMEM): F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA). For the in vitro evaluation of their cytotoxicity, the new derivatives were solubilized in DMSO at a concentration of 10 mg/mL and filtered with 0.22 µm Millex-GV Filter (Merck, Darmstadt, Germany). For cytotoxicity kinetics, the IncuCyte^® S3 Live-Cell Analysis System (Sartorius, AG, Goettingen, Germany) was used. Briefly, 2 × 10⁴ cells seeded in 96 well plates were treated with the new derivatives in a concentration ranging between 500 μg/mL and 3.9 μg/mL. The plates were placed in the IncuCyte^® S3 Live-Cell Analysis System, and five images per well were taken every six h over a 72-h period and then processed, according to kit recommendations.

Samples of PPy were extracted according to ISO 10993-12 with the following modification: 0.05 g PPy per 1 mL of cultivation medium was used instead of the ISO-defined ratio of 0.2 g polymer per 1 mL. The extraction was conducted in chemically inert closed containers using aseptic techniques at 37 ± 1 °C under stirring for 24 h. Subsequently, the extract was separated from the polymer powder by centrifugation at 1000 g for 15 min followed by the second centrifugation of supernatant under the same conditions. The parent extracts (100%) were used for the testing of cytotoxicity according to the ISO 10 993-5 protocol. The parent 100% extracts were diluted in complete medium to obtain a series of dilutions. All extracts were used within 24 h. Prior to in vitro testing, the extracts the extracts were sterilized by sterile filtration through a 0.22 μm Millex GV filter (Merck, Darmstadt, Germany). All tests were performed in four separate sets.

Radioactive ¹¹C was generated by the ¹⁴N(p, α)¹¹C nuclear reaction using a cyclotron. [¹¹C]CH₃I was prepared from [¹¹C]CO₂ according to the conventional method. The ¹¹C methylation of (2S,3S)-Desethylreboxetine to [¹¹C]MRB, HPLC purification and formulation were performed using an automated synthesis system (CUPID C-11-BII, Sumitomo Heavy Industries, Tokyo, Japan). Thus, obtained [¹¹C]CH₃I was trapped in 250 µL of anhydrous DMF containing 0.5 mg of (2S,3S)-Desethylreboxetine (1.75 µmol) and 8.5 µL of 5 M NaOH (42.5 µmol) at − 15 °C to − 20 °C, and then the reaction mixture was heated to 100 °C for 10 min. The radioactive mixture containing [¹¹C]MRB was diluted with 1 mL of HPLC mobile phase and transferred to a column (10 mm I.D. × 250 mm, CAPCELL PAK C18, SHISEIDO, Tokyo, Japan) attached to an HPLC system (JACSO, Tokyo, Japan). Elution with 30:70 v/v CH₃CN/0.2 M ammonium formate in sterile water at a flow rate of 5 mL/min yielded a radioactive fraction corresponding to pure [¹¹C]MRB (retention time: 9.3 min). The fraction was collected in a rotary evaporator and evaporated to dryness at approximately 90 °C under reduced pressure. The residue was dissolved in 3 mL of sterile tween-saline and filtered through a 0.22 µm Millex^Ⓡ-GV filter (Merck Millipore Ltd., USA). At the end of the synthesis, 0.5–1.3 GBq of [¹¹C]MRB was obtained with a molar activity of 34–102 GBq/µmol.