High-Throughput Analysis of Postmeiotic Male Germ Cells Using Image Flow Cytometry.

Given the considerable cellular heterogeneity in the testis, studies of male germ cell biology require robust techniques that can identify the changes taking place in each population of specific cell types. Imaging flow cytometry (IFC) has become a powerful tool for studying heterogeneous cell populations in high-content imaging mode. With considerable interest, because of its application to studies of male fertility, the question arises as to how we can accelerate its application to characterize and analyze sperm differentiation. We developed an IFC-based procedure to identify different spermatids stages and quantify the nuclear translocation of protamine 1 (PRM1), an important event during sperm differentiation. After tissue digestion with a protocol optimized for IFC, germ cell suspensions are stained for PRM1 and nucleus. Image analysis is performed using an optimized nuclear mask to detect DNA content, which allows for categorization of germ cells to differentiate spermatid stages. Next, a similarity score parameter to correlate the location of PRM1 staining and the nuclear dye is used to determine translocation of PRM1 from the cytoplasm to the nucleus. This technique measures PRM1 localization at a single cell level and can detect small changes in the subcellular distribution of PRM1 in selected cells, providing objective and robust quantitative data for statistical analysis. This protocol can be used, with slight modifications, to analyze the nuclear translocation of other proteins as well as in different species.