Tunneling nanotubes (TNT) are F-actin-based membranous tubes. They constitute a mechanism of communication and transfer of a variety of molecules and cell organelle. It was shown in previous studies that “artificial” TNTs formation tendency is increased in Gaucher cells. In this study, we worked on developing methods for the analysis of spontaneous TNTs formation of U937 monocyte-derived macrophages by fluorescent confocal microscopy.