In Vivo Tumor Microinjection Profiling

A20 cells (ATCC) were cultured in RPMI 1640 with l-Glutamine (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), and 50 nM β-Mercaptoethanol at 37 °C and 5% CO₂. All experiments in mice were approved by IACUC Board of Presage Biosciences, Seattle, WA (Protocol number PR-001) and were performed at Presage in accordance with relevant guidelines and regulations. For generating A20 allografts, female BALB/cAnNHsd mice (Envigo) were inoculated with 1 × 10⁶ A20 cells. CIVO IT microinjections were performed as described previously^{28 (link)}. Briefly, mice (n = 6 per time point, 4 and 24 h) were enrolled in microinjection studies when implanted tumors reached the following approximate dimensions: 14 mm (length), 10 mm (width) and 7 mm (depth). The CIVO device was configured with 6 thirty-gauge injection needles with a total volume delivery of 2 μL. Presage’s fluorescent tracking marker (FTM, 5% by volume) was added to the injection contents for spatial orientation. At 4 and 24 h following CIVO microinjections, mice were euthanized using CO₂ inhalation for biomarker analyses.