Hemavet hematology analyzer

LLC (5 x 10⁵) cells were inoculated subcutaneously in the right flank. The tumor volume was calculated using the formula V = lw² x 0.4 where l is the length and w the width [10 (link),11 (link),12 (link)]. Mice were monitored twice weekly by study participants at which time tumors were measured using calipers. Mice were euthanized prior to study endpoints if tumors were ulcerated or if the animals were moribund (obvious lack of grooming, extreme weight loss, signs of dehydration) or tumors were larger than 2500mm³. In this study, no animals were euthanized prior to study endpoints. Analgesic were not administered at any point during the study until euthanization. Blood was collected using EDTA-coated capillaries and analyzed with a Hemavet hematology analyzer (Drew Scientific). At sacrifice, blood was collected into 3.2% sodium citrate, centrifuged at 6000 rpm for plasma collection and centrifuged again at 12000 rpm to remove any contaminating cells. For brain collection, animals were perfused with 15ml of PBS under ketamine and xylazine anesthesia.

Femora and tibiae were dissected free into 2% FBS in PBS. Bone marrow cells (BMCs) were collected by centrifugation of mouse femora and tibiae at 6000–7000 g for 4 minutes at 4 °C. Cells were then incubated with red blood cell (RBC) lysis solution (BioLegend 420301) for 5 minutes on ice, washed once with BMC buffer, re-suspended in 0.5 mL of sterile BMC buffer, and passed through a 5-mL polystyrene round-bottom tube with a cell-strainer cap (Corning, 352235). One fourth of the spleen was mechanically dissociated with a razor blade, incubated with RBC lysis solution (BioLegend 420301) for 5 minutes on ice, washed once with PBS, and filtered through a 70-μM nylon mesh filter. Next, 200 μL of peripheral blood was incubated with RBC lysis solution (BioLegend 420301) for 10 minutes at room temperature, washed once with PBS passed through a 5-mL polystyrene round-bottom tube with a cell-strainer cap (Corning, 352235). At the experimental end points, blood was collected by intracardiac puncture with a 27-gauge needle into ethylenediaminetetraacetic acid (EDTA) Microtainer tubes (BD Pharmingen). Complete blood counts were obtained using a HEMAVET® hematology analyzer (Drew Scientific). Plasma was prepared by centrifugation of whole blood at 1500 g for 8 minutes at 4 °C.

White blood cell subpopulations (including lymphocytes, monocytes, and neutrophils) were quantified with a Hemavet hematology analyzer (Drew Scientific, Dallas, Texas, USA).