Microvette 500 k3 edta tubes

Experimental infections in mice were performed in biosafety level 3 (BSL-3) facilities at Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CISA, INIA-CSIC). Animals were handled in strict accordance with the guidelines of the European Community 86/609/CEE. A total of 59 six-week-old Hsd:ICR(CD-1) female mice (Envigo; Inotiv) were used (29 uninfected and 30 WNV-infected). Mice were infected intraperitoneally (i.p.) with 10⁴ plaque forming units (PFU) of WNV NY99 (GenBank: KC407666.1) in 200 µL of Minimum Essential Medium Eagle (Corning). Mock-infected animals were inoculated i.p. with the same volume of culture medium. Animals were kept with ad libitum access to food and water, and daily monitored for weight and clinical signs. At different days post-infection (dpi), a representative number of animals (8-10 mice per group) was anesthetized under isoflurane, and humanly sacrificed. Blood from submandibular vein, and left hemispheres of brain and cerebellum from euthanized mice, were collected at 3- (n = 9 uninfected and n = 10 infected), 7- (n = 10 uninfected and n = 10 infected), and 10-days post infection (dpi) (n = 10 uninfected and n = 8 infected). Plasma was separated using Microvette 500 K3 EDTA tubes (Sarstedt) by centrifugation (1123 × g, 15 min at 4°C). All samples were frozen at −80°C until analyses.

Retro‐orbital, mandibular or cardiac bleeds were collected in Microvette® 500 K3 EDTA tubes (Sarstedt, Nümbrecht, Germany), transferred to a 5‐mL polypropylene Falcon tube (Corning, New York, NY, USA), and adjusted to a total volume of 1 mL with phosphate‐buffered saline (PBS; Gibco, New York, NY, USA). The cell suspension was under‐layered with 2 mL of Histopaque‐1077 (Sigma Aldrich, St Louis, MO, USA) and centrifuged at 300 g for 15 min at room temperature (RT). Leukocytes can be found at the interface layer, these were then transferred to a 10‐mL Falcon tube, washed twice with ice‐cold PBS and resuspended in FACS buffer [PBS, 2% FBS (Bovogen Biologicals, Lot#2009A; Clayton, VIC, Australia), 1 mm ethylenediaminetetraacetic acid (EDTA; Invitrogen™, MA, USA)]. Enriched leukocytes were used in subsequent experiments.