Xt 2000iv

For the pharmacokinetic study, BALB/c mice were tail intravenously injected with 2G1 (15, 30, or 60 mg/kg), or equivalent volume of PBS. Three males and three females were in each subset. Blood samples were collected at 0.5, 6 h, 1, 2, 4, 7, 10, 15, 21, and 28 days after injection. Serum 2G1 concentration was quantified using ELISA. Briefly, mouse anti-human IgG Lambda (SouthBiotech) at 2 μg/mL was coated in ELISA plates. Serum samples and antibody 2G1 control were added into the plates and incubated for 1 h. After washing, a goat anti-human Fc HRP (Sigma) was used as secondary antibody with 1:6000 dilutions. After the chromogenic reaction by the HRP substrate (Solarbio), the plates were read at 450 nm.
Crlj:CD1(ICR) mice were randomly divided into control (treated with PBS), 15, 30, and 60 mg/kg groups for testing the in vivo toxicity of 2G1, with three males and three females each group. Body weight was tracked every 2 days. Blood samples were collected 14 days after administration and mice were subsequently euthanized for tissue damage detection. Blood indicators including white blood cell count, red blood cell count, hemoglobin, and platelets were measured in multiple automated hematology analyzer (Sysmex XT-2000iV). Pathological changes of hearts, livers, spleens, lungs and kidneys were examined by hematoxylin and eosin (H&E) staining.

All the blood coagulation-related values were measured by LSI Medience Corporation (Tokyo, Japan). APTT, PT, and fibrinogen level were measured with HemosIL SynthASil (Instrumentation Laboratory, Bedford, MA, US); HemosIL RecombiPlasTin 2G (Instrumentation Laboratory); and HemosIL Fibrinogen-C (Instrumentation Laboratory), respectively. These three variables were measured using ACL ELITE PRO (Instrumentation Laboratory). Platelet counts were measured with cellpack (Sysmex, Hyogo, Japan) using XT-2000iV (Sysmex).

Whole blood cell and platelet analysis were performed using an automated XT-2000iV veterinary hematology analyzer (Sysmex Europe GMBH, Norderstedt, Germany). Plasma cholesterol levels were measured by an enzymatic colorimetric assay (Roche diagnostics, Almere, The Netherlands). Protein C plasma levels were determined using an ELISA with a sheep anti-murine protein C polyclonal antibody (Haematologic Technologies Inc. Essex Junction (VT), USA), conjugated with Horseradish peroxidase with the EZ-Link Plus Activated Peroxidase Kit (Thermo Scientific, #31489, Waltham (MA), USA), as described previously^{41 (link)}. Fibrinogen and Serum Amyloid A plasma levels were determined using commercial ELISA kits (Affinity Biologicals, Ancaster (ON), Canada and R&D systems, Minneapolis (MN), USA, respectively). Plasma IL-6 levels were determined using a commercially available ELISA kit (BD Biosciences, San Jose CA, USA) following the manufactures instructions.

Blood samples were taken from the veins of animals at 6 and 12 months postsurgery. The blood samples were collected using a Sysmex XT‐2000iv automatic hematology analyzer (Sysmex, XT‐2000iv, Japan), centrifuged at 1000 g for 15 min and serum biochemical detection was performed using an automatic biochemical analyzer (Hitachi, 7180, Japan).

Lungs were lavaged with 1.5 ml of cold PBS, and the recovery rate of bronchoalveolar lavages fluid (BALF) was approximately 85%. Cells were isolated from the BALF by centrifugation at 300 g for 5 minutes at 4°C, and the cell pellets were resuspended in 500 μl of PBS; the obtained BALF samples were analyzed by using a flow‐cytometric hematology system (XT‐2000iV; Sysmex Corporation, Mundelein, IL, https://www.sysmex.com) to classify the number and type of inflammatory cells. The total cells in BALF were counted by a hemocytometer, and cell viability was measured by trypan blue exclusion.

Complete blood picture was determined using an automated blood cell analyzer (Sysmex XT-2000iV, Kobe, Japan). Blood glucose, total serum proteins, albumin (ALb), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Cr), calcium (Ca), phosphorus (P), and vitamin D were measured using commercial diagnostic kits obtained by Biomerieux, and Spinreact, Spain. Serum globulins were calculated mathematically by subtracting albumin from total proteins. Serum cortisol was measured using cortisol enzyme-linked immunosorbent assay kit [Biovision, USA (Cat. No. K7430-100)]. Malondialdehyde (MDA) was evaluated by ELISA kit (Cusabio Biotech Co. Ltd., China) using the methods of Ohkawa et al. [19 (link)]. Serum total antioxidant capacity (TAC) was determined using TAC assay kit [OxiSelect™, San Diego, CA, USA (Cat. No. STA-360)] according to Cerutti and Trump [20 ].