Ciprofloxacin

E. coli MG1655 (ATCC 700926) was used for all experiments. All cultures were routinely grown in LB medium and incubated at 37 °C with shaking at 180 rpm for liquid cultures. Ciprofloxacin (MP Biomedical) was added to the medium as indicated.

Extracts and pure compounds were tested for their antimicrobial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium intracellulare. The antifungal activities were evaluated against a panel of pathogenic fungi, including Candida albicans, C. glabrata, C. krusei, Aspergillus fumigatus, and Cryptococcus neoformans, associated with opportunistic infections. Ciprofloxacin (MP Biomedicals Inc, Aurora OH, USA) for antibacterial bioassays and Amphotericin B (MP Biomedicals Inc, Aurora OH, USA) for fungal bioassays were used as positive controls, respectively [45 (link)].

B. pseudomallei Bp82 (Propst et al., 2010 (link)), a ΔpurM mutant that is avirulent in immunocompetent and immunodeficient animals and is exempt from select-agent rules, was obtained from H. Schweizer. B. cenocepacia BC7, K56-2S, K56-2V (Varga et al., 2013 (link)) were obtained from J. Goldberg. B. multivorans CGD1 and CGD2 (Varga et al., 2012 (link)) were obtained from S. Holland. B. thailandensis E264 (Yu et al., 2006 (link)) was obtained from D. DeShazer. For Burkholderia, routine medium was Miller LB (Atlas, 2010 ) broth and agar. Pseudomonas aeruginosa PA14 (He et al., 2004 (link)) liquid medium was Miller LB (Atlas, 2010 ). Ciprofloxacin was obtained from MP Biomedicals (Aurora, Ohio, United States). Levofloxacin, Trimethoprim, Doxycycline, Gentamicin, and Cefotaxime were obtained from Sigma (St. Louis, MO). All bacteria were grown at 37°C in ambient air.

Dissected guts from E16.5 embryos were digested with an enzyme mix of 1 mg/mL Dispase/Collagenase (Roche, 296638), at 37 °C. To obtain a cell suspension, culture medium containing 500 μL OptiMEM (Life technologies, 11058-021), 10 % foetal calf serum (FCS, PAA Labs, A15-649), 1 % L-glutamine (Life technologies, 25030-024), 1 % penicillin/streptomycin (Invitrogen, 15140-122), and 0.1 % ciprofloxacin (MP Biomedicals LLC, 199020) antibiotics was added to the digested tissue to aid dissociation with manual pipetting. The suspension was centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded and the cell pellet was re-suspended in fresh culture medium (for short-term culture) or simple OptiMEM (for FACS).
For FACS, samples were purified in a FACS ARIA II cell sorter (BD, specialised personnel in Francis Crick Institute, Mill Hill) to isolate YFP⁺ cells. Isolated YFP⁺ cells were collected in OptiMEM and processed for RNA extraction and RT-PCR. For short-term culture, cell suspensions were plated onto Fibronectin (Sigma, F1141)-coated, 8-well Lab-Tek permanox chamber (Thermo Fisher Scientific, 177445) slides. Cultures were then allowed to settle for approximately 15 hours in a 5 % CO₂ incubator before being fixed and processed for immunofluorescence.

PC3, HepG2, and MDA-MB-231 cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM (Sigma-Aldrich, Helsinki, Finland), supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany), 1% non-essential amino acids (Sigma-Aldrich), and 0.5% ciprofloxacin (MP Biomedicals, Illkirch Cedex, France), at 37 °C in an atmosphere of 16% O2, 5% CO2, 79% N2 and 97% humidity. Transient transfections and luciferase assays were performed as described [29 (link)].