Something old, something new, something borrowed, something green

ALMOST 30 years have passed since Nolan et al. published the breakthrough manuscript on the development of the FACS-Gal detection system (1), a sensitive assay for the analysis of the expression of the Escherichia coli lacZ reporter gene combined with a gene of interest. The methods involve the flow cytometric analysis and cell sorting of viable cells based on the levels of activity of the b-galactosidase (b-gal) enzyme encoded by this gene. Three years later, a significant improvement of the original FACS-Gal assay was published by the same group of authors (2) bringing new information and advanced analyses that added more strength and reproducibility to this powerful assay used to study gene expression in eukaryotic cells. In parallel, the reporter gene technology has been enriched by the discovery, cloning and development of the green fluorescent protein (GFP) and a number of GFP variants (3–6). All these developments enabled researchers to study a variety of cellular processes both in vitro and in vivo/ ex vivo. The article of Sanchez-Luengo et al. (this issue, page 721) represents a further progress in the technical aspects of the FACS-Gal reporter system, investigating lung and haematopoietic tissues of mice. Given that a successful readout depends on the b-galactosidase substrate, namely di-b-D-galactopyranoside (FDG), the authors have optimised the assay by performing a broad examination of the FDG loading conditions, including the timing of its administration and the tissue/cell type to which it is delivered, as well as how it is influenced by the method of choice for tissue dissociation. Isolation of various cell types from different organs demands the use of mechanical and/or enzymatic approaches for tissue disaggregation, followed by a manual and/or automated method to obtain the final single-cell suspension. Researchers nowadays use various tools for the combined mechanical/enzymatic tissue disaggregation such as the Miltenyi GentleMACS, the Becton Dickinson Medimachine or the Bertin Instruments Precellys Evolution. Regarding the mechanical protocol of the article by Sanchez-Luengo et al., spleen and lungs were minced and disintegrated over a 40 mm cell strainer followed by erythrocyte lysis with ammonium chloride; lungs were then disintegrated using the GentleMacs device. For the combined mechanical/enzymatic dissociation of spleen, thymus and lungs, the GentleMacs technology was used. A significant difference of the cell viability was observed between the mechanical and mechanical/enzymatic method for isolation of lung cells. Pneumocytes contribution to the total cell population was decreased significantly when lungs were disintegrated using the mechanical protocol only. The authors presumed that this was a consequence of pneumocytes not being properly released from the extracellular matrix, which certainly holds true. However, we believe it would be interesting to compare and analyze the list mode data files from the analysis of solely mechanical versus the mechanical/enzymatic protocol using the back gating procedure all over the applied gating strategy to see the distribution of each of the determined cell subpopulation on various combinations of scatter and fluorescence parameters. Moreover, the debris ratio can be very high in such samples and, even worse, larger pieces of debris from extracellular matrix are often as large as cells, making it impossible to completely exclude the debris from the cells based on the scatter parameters only. Since the authors used a powerful four-laser