Common use of Heart Clause in Contracts

Heart. Mice were anaesthetised with isoflurane, and exsanguination was achieved by drawing blood from the abdominal IVC. The rib cage was then opened to reveal the heart. The heart was perfused with 1ml cold sterile saline through the aorta and removed from the animal. The heart was placed in a 40mm petri dish and sliced in half using a blade. Any remaining blood was flushed from the ventricles using saline, and the tissue placed in a fresh petri dish containing 1ml heart digestion media. The tissue was minced to 1mm pieces using microscissors and transferred to a 5ml polypropylene tube, which was placed on ice. ▇▇▇▇▇▇ was allowed to settle at the bottom of the tube and supernatant aspirated to a 50ml Falcon tube with 100µm nylon mesh attached. The mesh was washed with heart digestion media, and the tube placed on ice. To the heart tissue, 1ml digestion mix (heart digestion media plus 10µg/ml type 1 collagenase (Sigma-▇▇▇▇▇▇▇; Merck KGaA) and 10µg/ml DNAse 1) pre-warmed to 37ºC was added, and the tube incubated at 37ºC for 15 minutes. The tube was vortexed for 1 minute half way through the incubation. This process was repeated, and after the final incubation the remaining undigested tissue was mashed through a 100µm nylon mesh with the plunger of a 1ml syringe. The tube containing the digested heart tissue was centrifuged at 1100RPM for 5 minutes at 4ºC. The supernatant was discarded and the tissue re-suspended in 3ml 0.01M EDTA in FCS at room temperature. The suspension was then layered over 3ml Histopaque-1077 (Sigma-▇▇▇▇▇▇▇; Merck KGaA) at room temperature, and centrifuged for 30 minutes at 900RPM at room temperature with no brake. Leukocytes form an opaque layer between the Histopaque-1077 and the FCS. Due to low yields of leukocytes from heart tissue, the FCS and leukocyte layer were carefully removed to a 15ml Falcon tube and topped up to 10ml with PBS. The tube was spun at 1100RPM for 10 minutes at 4ºC. The supernatant was discarded and the heart leukocytes were re-suspended in FACS buffer.