Filter sample bags

Canine whole blood was collected in heparin sulfate tubes from beagle dogs as well as dogs from mixed breeds. Whole blood (20 μL) was plated in 96-well plates (Corning Costar, Tewksbury, MA, USA) with 180 μL of DMEM complete medium (Dulbecco's modified Eagle medium; 10% heat-inactivated fetal bovine serum; 100 U/mL penicillin; 100 μg/mL streptomycin; Gibco Life Technologies, Grand Island, NY, USA) containing vehicle control or oclacitinib (0.001–10 μm), concanavalin A (ConA; 1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), and canine interleukin-2 (IL-2; 50 ng/mL; R&D Systems, Minneapolis, MN, USA). Plates were incubated at 37 °C for 48 h. Tritiated thymidine, 0.4 μCi per well (Perkin Elmer, Waltham, MA, USA), was added for 20 additional hours. Plates were frozen and then thawed, washed, and filtered using a Brandel MLR-96 cell harvester (Gaithersburg, MD, USA) and prewet filter mats (Perkin Elmer). Filters were dried and placed into filter sample bags (Perkin Elmer) with 10 mL of scintillant (Perkin Elmer). Sealed filters were counted on an LKB Wallac 1205 Betaplate liquid scintillation counter (Pharmacia, Uppsala, Sweden). Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.

Canine bone marrow cells were isolated under sterile conditions from the humerus of beagle dogs and placed in warmed DMEM medium (Gibco Life Technologies). The sample was filtered through a stainless steel wire mesh (Sigma-Aldrich) and gently ground with a glass pestle. The suspension was then passed through a 100-μm cell strainer and washed three times in warmed Alsever's solution (Sigma-Aldrich). The final pellet was resuspended in 20 mL of DMEM complete medium. Cell suspensions were plated in 96-well plates (Costar Corning) at a density of 2 × 10⁵ cells per well in DMEM complete medium containing oclacitinib (0.001–10 μm) or vehicle control, and Erythropoietin (0.2 U per well Erythropoietin; R&D Systems). Plates were incubated at 37 °C for 48 h. Tritiated thymidine, 0.4 μCi per well (Perkin Elmer), was added for 20 additional hours. Plates were frozen and then thawed, washed, and filtered using a Brandel MLR-96 cell harvester and prewet filter mats (Perkin Elmer). Filters were dried at 60 °C for 1 h and placed into filter sample bags (Perkin Elmer) with 10 mL of scintillant (Perkin Elmer). Sealed filters were counted on an LKB Wallac 1205 Betaplate liquid scintillation counter. Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.