Lithium heparin microtainers

Eastern box turtles (EBT) were collected from five field sites in Illinois (Collison, Forest Glen, Kennekuk, Kickapoo, and Forbes) using dog-assisted capture as previously described (10 ). Turtles were weighed, assigned to an age class (adults were >200 g, juveniles were <200 g), and sexed using a combination of sexually dimorphic traits (11 ). Complete physical examinations were conducted by a single observer (LA). Turtles with clinical signs of illness (ocular/nasal discharge, oral plaques, open-mouth breathing, etc.) or active injuries were categorized as “unhealthy,” while turtles with no clinical signs of illness or fully-healed injuries were considered “apparently healthy.”
Blood (<0.08% body weight) was collected via the subcarapacial sinus, immediately placed into lithium heparin microtainers (Becton, Dickinson and Company, 1 Becton Drive, Franklin Lakes, NJ), and stored on ice packs until processing (2–6 h after collection). Blood that was obviously lymph contaminated or clotted was not used in analyses. All activities involving live animals were approved by the University of Illinois Institutional Animal Care and Use Committee (Protocol 18000).

Turtles were examined twice daily (morning, evening) for clinical signs related to ranaviral disease, including: ocular discharge, nasal discharge, oral plaques, palpebral swelling, cutaneous abscesses, lethargy, and injection site swelling. All turtles were handled for a maximum of two minutes, and then returned to their enclosure on their basking bricks. Gloves were changed after handling each turtle.
Twice weekly, turtles were weighed, and venous blood (3 ml) was drawn from the subcarapacial sinus. Blood was immediately aliquoted in two separate lithium heparin microtainers (Becton-Dickinson, Franklin Lakes, NJ) and kept on ice until processing 1 hour later.

In rats, whole blood was collected in lithium heparin microtainers (BD) and plasma isolated via centrifugation. In NHPs, whole blood was collected in SST vacutainers (BD) and serum isolated via centrifugation. Tissues were homogenized using T-PER buffer (Thermo Scientific) supplemented with Protease Inhibitor Tablets (Thermo Scientific). CTLA4Ig was quantified using CTLA-4 Soluble Human ELISA Kit (Invitrogen); ALS was quantified with Easy-titer Rabbit IgG (Thermo Scientific); ATG was quantified with Rabbit Fc kit (Perkin Elmer); and anti-CD154 was quantified with Human IgG Kappa MAb kit (Perkin Elmer).