Calcein and its derivative calcein AM are generally supplied as a lyophilized solid and must be resuspended in a solution, usually DMSO, before loading into cells. To make a calcein or calcein AM stock solution, reconstitute the dye in high-quality, anhydrous DMSO, such that the concentration is approximately 1 mg/mL. For example, 50 µL of DMSO added to 50 µg of calcein AM will yield a 1 mg/mL stock solution. For calcein AM, further dilution of the stock solution into an aqueous buffer (e.g., HBSS) is required before loading into cells. As an aqueous solution, calcein AM is susceptible to hydrolysis and should be used within one day.