Net027a001mc

cMuStem cells (n = 7 independent cell batches) were thawed and seeded at 1 × 10⁴ cells/cm² for 1 week. Next, host PBMCs harvested before and after cell delivery were thawed in cMuStem cell growth medium and cultured overnight at 37°C and 5% CO₂. cMuStem cells were harvested and, together with splenocytes taken from another dog and used as a positive control, were irradiated at 35 Gy. PBMCs were then cultured in quadruplicate at 1 × 10⁵ cells per well in 96-well plates with irradiated cMuStem cells. In parallel, PBMCs were also cultured with control splenocytes or stimulated with 10 μg/mL Concanavalin A (ConA; Sigma-Aldrich) to serve as a positive control of proliferation. cMuStem cells were diluted at 2:1, 1:1, 1:4, 1:16 and 1:64 (MuStem cells: PBMCs) to demonstrate a dose effect. After 5 days of co-culture, cells were incubated overnight with tritiated thymidine (1:40; NET027A001MC, Perkin Elmer, Waltham, MA, USA) at 37°C and 5% CO₂. Cells were harvested on a filter using a Perkin Elmer Harvester and radioactivity was measured as counts per minute (cpm) using a MicroBeta plate counter (Perkin Elmer).