Liberase blendzyme

When the tumors reached a mean volume >400 mm³, the mice were randomized into four study groups: PBS + vehicle, PBS + alisertib, HSV1716 + vehicle and HSV1716 + alisertib. A single dose of PBS or HSV1716 was injected intratumorally on day 0 followed by oral gavage administration of vehicle or alisertib at 24 hours and 48 hours post infection. Tumors and spleens were harvested at 72 hours post infection and then prepared for flow cytometric analysis. Briefly, fresh whole spleens were transferred to 2 mL PBS and then smashed through a 70 μM cell strainer with 5 mL syringe plunger. Whole tumors were transferred to 6-well dishes containing 2 mL PBS and then processed as previously described [56 (link)]. Briefly, tumors were minced by mechanical chopping and incubated in PBS containing 25 μg/mL liberase blendzyme (Roche Diagnostics., Indianapolis, IN) and 150 μg/mL DNAse I for 1 hour at 37°C. Tumor samples were then passed through a 70 μM cell strainer with the aid of a 5 mL syringe plunger. After washing splenocytes and tumor cell suspensions once with PBS, the cell suspensions were cleared of red blood cells (RBCs) with ACK lysing buffer (Lonza, Walkersville, MD). The cells were then blocked with 5% mouse Fc blocking reagent (Miltenyi Biotec Inc., San Diego, CA) in FACS buffer (1% FBS and 1mM EDTA in PBS) and analyzed by flow cytometry.

MEM, FBS, and penicillin-streptomycin were purchased from Life Technologies. Liberase Blendzyme and collagenase were from Roche Molecular Biochemicals, and trypsin was from Worthington. α-conopeptides Vc1.1 and RgIA were synthesized as reported previously (Cartier et al., 1996 (link); Ellison et al., 2008 (link)). All other chemicals were obtained from Sigma-Aldrich.

Wild-type and genetically modified C57BL/6 mice (10–12 weeks old) were used for cardiomyocyte preparation. Genetically modified mice carried a heterozygous (+/-) or a homozygous deletion (-/-) of the KChIP2 gene. The generation and reactivation of the KChIP2 knockout mouse line have been previously described [9 (link), 21 (link)]. For our experiments the animals were obtained from the local animal facility, where they were held at room temperature (20–22°C), with food and water available ad libitum, and where they showed normal breeding. Genotyping was done based on tail biopsy. Mice were sacrificed by cervical dislocation under isoflurane anesthesia. The heart was excised, mounted on a temperature-controlled (37°C) Langendorff perfusion system and rinsed with a Ca²⁺-free solution (113 mM NaCl, 4.7 mM KCl, 0.6 mM KH₂PO₄, 0.6 mM Na₂HPO₄, 1.2 mM MgSO₄, 12 mM NaHCO₃, 10 mM KHCO₃, 30 mM taurine, 5.5 mM glucose, 10 mM HEPES, pH 7.46). Then the heart was digested for 9–10 min with the same solution containing 0.1 mg/ml Liberase Blendzyme (Roche). Epicardial tissue was obtained from the left ventricular free wall and dissociated into single cells. Ca²⁺ was stepwise reintroduced to a final concentration of 0.5–1 mM.