Sterile 96 well plate

The cover of a sterile 96-well plate (Corning/Costar, New York, NY, USA) was used for specimen preparation [42 (link)]. A total volume of 30 μL of the adhesives was added to the round indentions on the cover with a micropipette (Eppendorf, Hamburg, Germany). All visible air bubbles were carefully removed using a hypodermic needle (Weigao Group Medical Polymer Co., Ltd., Weihai, China). The adhesive was photopolymerized for 10 seconds (s) by a light-emitting diode (LED) curing light (Elipar S10; 3M ESPE) with a polyester strip covering to obtain a specimen disk of 8 mm in diameter and 0.5 mm thick. The power density of the light was maintained at 1200 mW/cm². All specimens were immersed in distilled water at 37 °C and agitated constantly with a magnetic stirrer (CJJ 78-1; Shuai Deng Instrument Co., Ltd., Shanghai, China) for 1 hour (h) to remove unpolymerized monomers [43 (link)]. The obtained disks were dried at room temperature and then were sterilized using ultraviolet light for 1 h [34 (link)].

The sub-inhibitory concentration (SIC) of garlic and ginger oil was determined according to a previously published protocol [10 (link)]. Two-fold dilutions of garlic and ginger oil (0, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039%) were prepared in a sterile 96-well plate (Costar, Corning, NY, USA) containing 100 μL of TSB broth. Ethanol was also included at the concentrations found in the corresponding garlic and ginger samples. Wells were subsequently inoculated with 100 μL of S. infantis, S. typhimurium, or S. enteritidis (~10⁶ CFU/mL). Plates were placed in a Cytation 5 multimode microplate reader (Agilent, Santa Clara, CA, USA) and incubated at 37 °C for 24 h with OD600 readings taken every 30 min. Following incubation, samples were diluted, plated onto XLD plates, and incubated for 24 h at 37 °C for Salmonella enumeration. All concentrations were run in duplicate, and experiments were repeated three times for each Salmonella strain. The highest concentration of phytochemicals that did not inhibit Salmonella growth was selected as the SIC and used in subsequent experiments.

Dilutions of the antibody of interest were made in a sterile 96 well plate (Costar/Corning Incorporated, Corning, NY) in Eagle Minimum Essential Media (EMEM) (Sigma Aldrich, St. Louis, MO) supplemented with 5% FBS. In sterile 6 well plate (Costar/Corning Incorporated, Corning, NY), 125 μL of authentic EBOV or SUDV diluted to 1200 pfu/mL were added to each well and the plates were incubated at 37 °C for 1 hour. Virus was added to a well containing media alone (no antibody) as a control for 100% infection. Vero-E6 cells were exposed to 100 μL of the virus/antibody mixture and incubated at 37 °C for an additional hour. During this time, the plates were gently rocked every 15 minutes to ensure homogeneity and prevent drying. After 1 hour of incubation, 2 mLs of primary overlay (EMEM with 10% FBS and 1% Gentamicin (Sigma Aldrich, St. Louis, MO) with 1% SeaKem ME agarose (Lonza, Cohasset, MN)) was added to each well and the plates were incubated at 37 °C for 6 days. On Day 7 post-exposure to virus, neutral red solution (EMEM with 10% FBS and 1% Gentamicin with 5% neutral red (Gibco/Invitrogen, Grand Island, NY)) was added to all cell-containing wells and cells were incubated at 37 °C overnight. Infection was scored by counting the number of plaques per well, using the number of plaques on the control well (no antibody) as 100% infection.