Sperm Intracellular Calcium Evaluation.

The evaluation of the intracellular calcium is essential in studying sperm capacitation and acrosome reaction, among other processes. Here, we describe the use of chlortetracycline (CTC) to study Ca2+ distribution in relation to the sperm capacitation state, and the evaluation of Ca2+ levels in spermatozoa using a low or a high-affinity Ca2+ dye (Rhod-5 N-AM and Fluo-4-AM, respectively) by flow cytometry. Rhod-5 N-AM staining method also allows the study of Ca2+ in subcellular compartments, whereas the changes in intracellular Ca2+ levels can be measured in spermatozoa loaded with Fluo-4-AM by plate reader fluorometry.