Sterile syringe

Vials containing 1 mL CF33-hNIS-anti-PD-L1 virus (lab stocks) at a concentration of 1E08 or 1E06 PFU/mL were taken out of the −80°C freezer and thawed at room temperature. The stocks were then diluted 1:100 with PBS to obtain 1E06 or 1E04 PFU/mL. Next, 10 mL of diluted viruses were loaded in 10 mL sterile syringes (catalog #309604, lot #6291821; BD Biosciences) fitted with sterile 25G needles (catalog #305125, lot #0274523; BD Biosciences). The plunger of the syringe was slowly pushed to eject virus solution from the syringe, and the ejected viruses were collected in a 15 mL conical tube and were immediately used for titration by plaque assay.

JBCNJ055, EDCB0283, EDCB093, and JBCND053 (Selection based on distinct toxin profiles or sources) were used in cell free trials. 100 μl of overnight culture, of each isolate was inoculated in 5 mL of FTG (Oxoid, United Kingdom) and incubated at 37°C for 5 h. The culture was centrifuged at 3170 ×g (Heraeus Megafuge 8) for 3 min and 1 mL of the supernatant was transferred to a 1.5 mL microcentrifuge tube (Eppendorf, United Kingdom). The remaining supernatant was removed from the bacterial pellet and discarded. The pellet was resuspended in 3 mL of sterile FTG (Oxoid, United Kingdom) and 1 mL of the resuspended culture transferred to a 1.5 mL microcentrifuge tube. The contents of each microcentrifuge tube were drawn into separate 1 mL sterile syringes (BD Plastipak) and fitted with a 30 g needle. Ten larvae were challenged with 10 uL of each inoculate, from each isolate and were placed in sterile Petri-dishes lined with greaseproof paper (for better contrast in images). The plates were incubated at 37°C for 72 h. Survival percentages were recorded, and each experiment repeated in triplicate.

NTA measurements were performed with a NanoSight LM20 (NanoSight, Amesbury, United Kingdom), equipped with a sample chamber with a 640-nm laser. The samples were injected into the sample chamber with sterile syringes (BD Discardit II, New Jersey, USA) until the liquid reached the tip of the nozzle. All measurements were performed at room temperature. The software used for capturing and analyzing the data was the NTA 2.0 Build 127. The values obtained were concentration (particles/ml) and size and these were plotted in SigmaPlot 12.0 software.

Five-milliliter sterile syringes (BD), gavage needles (16-gage, 80 mm, curved needle), 1.5-ml/2-ml centrifuge tubes (Axygen, United States), 50-ml/15-ml centrifuge tubes (Corning, United States), 96-well cell culture plates (Corning, United States), 10 kD filters (Pall, United States), Oasis HLB solid phase extraction column (Waters, United States), 1-ml/200-μl/20-μl pipette tips (Axygen, United States), a BCA kit (Thermo Fisher Scientific, United States), high pH reverse peptide separation kit (Thermo Fisher Scientific, United States), and iRT (indexed retention time, BioGnosis, United Kingdom) were used.