β-Casomorphin, human (Human β-casomorphin 7)

Cell experiment:

To study the effect of β-Casomorphin, human and DAMGO, cells are seeded in 12-well culture plates. Experiments are performed 3 (DHE cells) or 21 days (HT29-MTX cells) after confluency. Twenty-four hours before the studies, the culture medium is replaced by serum-free medium to starve the cells from serum and to eliminate any interference from extraneous proteins or hormones. The experimental protocol is then the following: the serum-free medium is removed, and the monolayer cultures of DHE or HT29-MTX are washed twice with PBS (37°C). Serum-free medium with or without β-Casomorphin, human or DAMGO is added to the cells and incubated at 37°C for 30 min to 24 h in a humidified atmosphere. μ-Opioid receptor blockade is performed by preincubating the cells with cyprodime for 30 min before agonist addition. The supernatants are then collected, frozen, and stored at 20°C. Cells are processed with trypsin. The cell numbers per well are determined, and total RNA is isolated. All experiments are performed at least three times in triplicate[2].

References:

[1]. Brantl V. Novel opioid peptides derived from human beta-casein: human beta-casomorphins. Eur J Pharmacol. 1984 Oct 30;106(1):213-4.
[2]. Koch G, et al. Opioid activities of human beta-casomorphins. Naunyn Schmiedebergs Arch Pharmacol. 1985 Dec;331(4):351-4.