Cell experiment:

Coelomocytes suspensions (ISO and CF) are separately incubated with calcein-AM (excitation and emission wavelength: 496 nm and 516 nm, respectively) at a final concentration of 200 nM, during 30 min, at 26℃. Calcein-AM is a nonfluorescent ABC transporter substrate whose intracellular accumulation is inversely proportional to ABC transporter activity. Intracellular esterases convert calcein-AM into the fluorescent dye calcein, which is not an ABC transporter substrate, thereby accumulating the dye inside the cell. Therefore, a high fluorescence signal indicates low ABC transporter activity whereas a low fluorescence signal indicates high activity. The fluorescence of samples is measured by flow cytometer. The experiment is repeated six times in duplicates.

Calcein is a fluorescent dye and self-quenching probe, used as an indicator of lipid vesicle leakage, and also as a complexometric indicator for titration of calcium ions with EDTA, and for fluorometric determination of calcium.

Calcein accumulates intracellularly in coelomocytes incubated in coelomic fluid or ISO-EDTA. Coelomocytes incubated in CF show an increase in the calcein fluorescence intensity compared to the control.

[1]. Marques-Santos LF, et al. ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas. Fish Shellfish Immunol. 2017 Sep 5;70:195-203.