Procyte dx hematology analyzer

All neonatal CBCs were performed on pups between 3 and 4 days old. Blood was collected from the trunk via decapitation. Total red blood cells, WBCs, neutrophils, lymphocytes, monocytes, eosinophils and basophils were quantified by both automated (IDEXX ProCyte Dx Hematology Analyzer) and manual counts.

Cytokine/chemokine expression profiles were analyzed using a customized bead array (IDEXX ProCyte Dx Hematology Analyzer) to measure IL-6, IL-8, IL-10, CXCL1, CXCL2, CCL5, and CCL2. All measurements were conducted in a blinded manner.

Blood samples used for all measurements were collected from the cephalic veins prior to any treatments. Blood samples for complete blood counts were collected in polypropylene tubes containing 1.6 mg ethylenediamine tetraacetic acid/mL blood (Fa. Sarstedt AG & Co., Nümbrecht, Germany) and analysed using a ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Inc., Westbrook, ME, USA), which counted red blood cells, white blood cells, haemoglobin and platelets, among other things. Some of these values were also used for subsequent analyses related to our measured serum Ki-67 concentrations, which are discussed in more detail below.
Standard serum tubes (Fa. Sarstedt AG & Co., Nümbrecht, Germany) were used to take serum samples for serum chemistry and then centrifuged in an Eppendorf centrifuge 5424 (Fa. Eppendorf AG, Hamburg, Germany) at 3000× g for 5 min. The serum was then removed from the tube and analysed using the clinical chemistry analyser (Konelab 20i; Fa. Thermo Fischer Scientific Inc., Dreieich, Germany) and commercial kits according to standard procedures.
The serum for Ki-67 measurements was allowed to rest at room temperature for 2 h and then centrifuged at 1000× g for 15 min. It was then pipetted into Eppendorf tubes, frozen at −80 °C and stored under these conditions until processed within the next 3 months.

For serum chemistry, blood was collected into tubes containing a serum separator, the tubes were centrifuged, and the serum was obtained for analysis. Serum chemistry was performed on a Beckman Coulter AU680 analyzer, and the concentration of the following analytes was determined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine kinase, gamma-glutamyl transpeptidase, albumin, total protein, globulin, total bilirubin, blood urea nitrogen, creatinine, cholesterol, triglycerides, glucose, calcium, phosphorus, chloride, potassium, and sodium. Na/K ratio and albumin/globulin ratio were calculated. For hematology, blood was collected retro-orbitally into EDTA microtainers. Automated analysis was performed on an IDEXX Procyte DX hematology analyzer.

Blood was collected at scheduled time points (Days −7, 0, 3, 5, 7, 9, 11, 13, and 14, relative to exposure). Coagulation times were determined on whole blood collected with no additives, using an IDEXX Coag Dx Analyzer (IDEXX Laboratories, Westbrook, ME, USA). These blood samples were processed to obtain the serum. Serum was analyzed to measure C-reactive protein (CRP) levels using a Piccolo BioChemistry Panel Plus on a Vet Scan analyzer (Abaxis, Inc., Union City, CA, USA). Whole blood was also collected into tubes containing ethylenediaminetetraacetic acid (EDTA) for complete blood counts (CBC) using a Procyte Dx Hematology Analyzer (IDEXX laboratories, Westbrook, ME, USA) and for clinical chemistry using the mammalian liver enzyme profile rotor on a Vet Scan analyzer (Abaxis, Inc., Union City, CA, USA).

Animals were euthanized using an overdose of sodium pentobarbital (200–300 mg/kg, Vortech; Dearborn, MI) administered by intraperitoneal injection, followed by exsanguination. Weight, nose-to-tail length, and abdominal girth were measured, and body mass index (BMI) was calculated. Blood was collected from the vena cava in EDTA-treated collection tubes and cells were analyzed using a ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Inc.; Norcross, GA). Blood used for serum samples was collected in BD Vacutainer™ Serum Separation Tubes, left at room temperature for 1.5 h, and centrifuged at 25,000 g for 20 min. Fresh serum was used for measurements of high-density lipoprotein (HDL) (#ab65390, Abcam, Cambridge, MA) and blood chemistry (Catalyst Dx Chemistry Analyzer, IDEXX Laboratories, Inc). Epididymal fat pads were removed and weighed. Serum samples were stored at -80 °C until used for ELISA and cytokine analysis. ELISAs were conducted following the manufacturers’ protocols: leptin (#MOB00B, R&D Systems, Minneapolis, MN) and adiponectin (#Acrp30, R&D Systems). Insulin was measured using the Ultra-Sensitive Rat Insulin Elisa Kit (#90060, Crystal Chem; Elk Grove Village, IL). Serum cytokines were measured using the MSD V-PLEX Proinflammatory Panel 2 (rat) kit and MESO QuickPlex SQ 120 (Meso Scale Diagnostics; Rockville, MD).