Molecular imaging is the future of personalized medicine; however, it requires effective contrast agents. Hyperpolarized chemical exchange saturation transfer (HyperCEST) can boost the signal of Hyperpolarized 129Xe MRI and render it a molecular imaging modality of high efficiency. Cucurbit[6]uril (CB6) has been successfully employed in vivo as a contrast agent for HyperCEST MRI, however its performance in a clinical MRI scanner has yet to be optimized. In this study, MRI pulse sequence parameter optimization was first performed in CB6 solutions in phosphate-buffered saline (PBS), and subsequently in whole sterile citrated bovine blood. The performance of four different depolarization pulse shapes (sinusoidal, 3-lobe sinc (3LS), rectangular (block), and hyperbolic secant (hypsec) was optimized. The detectability limits of CB6 in a clinical 3.0T MRI scanner was assessed using the optimized pulse sequences. The 3LS depolarization pulses performed best, and demonstrated 24% depletion in a 25µM solution of CB6 in PBS. It performed similarly in blood. The CB6 detectability limit was found to be 100µM in citrated bovine blood with a correspondent HyperCEST depletion of 30% ± 9%. For the first time, the HP 129Xe HyperCEST effect was observed in red blood cells (RBC) and had a similar strength as HyperCEST in plasma.