Vetscan hm5 hematology analyzer

For the hematology study of anti-RBC sdAbs RMC1 and RB12, Swiss Webster mice were dosed at 5 mg/kg via retro-orbital injection (n = 5). Blood was sampled through retro-orbital collection prior to dosing for the determination of baseline measurements of hematological parameters. Additional samples were obtained at several time-points up to 28 days post-dosing. Blood samples were collected using EDTA-coated capillary pipette tubes. Red blood cell concentration, hemoglobin concentration, and hematocrit were determined using a VetScan HM5 Hematology Analyzer (Abaxis Inc., Union City, CA, USA), normalized by the baseline measurements, and reported as a percentage of pretreatment values. In addition to hematological parameters, body weight change was also monitored and analyzed.

To evaluate the possible immunomodulatory effects of PHA 568487 in vivo, the air pouch model of acute inflammation was used [12 (link),25 (link)]. At 8 weeks of age, male C57BL/6JRj mice (Janvier Labs, Le Genest-Saint-Isle, France) were injected with 3 mL of sterile air between the scapulae under isoflurane anesthesia. Three days later, the air pouch was re-filled with 3 mL of sterile air. On the sixth day, the mice received an intraperitoneal injection of either PBS (Thermo Fisher Scientific, Waltham, MA, USA) or PHA 568487 (Tocris Bioscience, Ellisville, MO, USA) at 12.4 μmol kg⁻¹ (5 mg/kg) or 123.4 μmol kg⁻¹ (50 mg/kg). Fifteen minutes later, all mice were injected with LPS (10 μg, List Biological Laboratories Inc., Campbell, CA, USA) directly into the pouch. The mice were sacrificed six hours after the LPS injection by an overdose of pentobarbital (Apoteket AB, Stockholm, Sweden). Finally, the pouch cavity was flushed with 2 mL of PBS with 1.6 mmol L⁻¹ of EDTA, and the exudate was collected. The white blood cell count of the exudate was analyzed using the Vetscan HM5 Hematology Analyzer (Abaxis, Union City, CA, USA) and thereafter centrifuged for 5 min at 400× g. Supernatants were stored at −80 °C until multiplex analysis.