Measurement of Pulsatile Hormone Release from Perifused Pituitary Cells Immobilized on Microcarriers

Abstract

This chapter discusses a method for measurement of pulsatile hormone release from perifused pituitary cells immobilized on microcarriers. Following cervical dislocation and decapitation, pituitaries are removed from weanling female Sprague-Dawley rats and collected in a 50-ml conical centrifuge tube containing fresh, sterile media (M199/BSA). The pituitaries are rinsed several times with M199/BSA and placed in a Petri dish, where each pituitary is cut into 6–8 pieces using a sterile razor blade. The pituitary pieces are allowed to settle in fresh M199/BSA in a sterile centrifuge tube. The M199/BSA is decanted and replaced twice with fresh M199/BSA to remove lysed cells and their products. After centrifugation of the cell filtrate at 225 g for 10 min at 23°C, the supernatant is discarded and the pellet is resuspended in culture medium. A cell suspension aliquot of 0.55 ml is delivered to each cell chamber. Culture medium is added to bring the final volume of each cell chamber to 1.0 ml. The cell chambers are gently shaken to mix the cell suspension and the microcarriers. Cell viability, which is assessed by Trypan blue dye exclusion, is typically in excess of 95% at the completion of the dispersion.