Scanning flow cytometers represent an appealing alternative to convention flow cytometers as they exploit measurements of the angle-resolved scattered light to provide accurate and quantitative estimates of cellular properties, but the requirements of current setups are unsuitable for integration with other lab-on-chip technologies or for point-of-care applications. Here we present the first microfluidic scanning flow cytometer (μSFC), able to achieve accurate angle-resolved scattering measurements within a standard polydimethylsiloxane microfluidic chip. 

Reale R., Peruzzi G., Ghoreishi M., Stabile H., Ruocco G., Leonetti M. (2023). A low-cost, label-free microfluidic scanning flow cytometer for high-accuracy quantification of size and refractive index of particles, Lab on a Chip - Miniaturisation for Chemistry and Biology.

Phenotypic heterogeneity of cell populations recently gained much attention. This phenomenon can arise from variability of nongenetic origin or fluctuations occurring during cell division of stochastically partitioned molecules between the two daughter cells, but discrimination of the two contributions is quite difficult. To systematically analyze these problems, we devised an experimental and computational apparatus to follow cell populations proliferation and directly measure how different cellular elements partition. Our method is based on staining different cellular components with fluorescent markers at the beginning, sorting the stained cell population, and then measuring the dynamics of the partitioning process.

Peruzzi G., Miotto M., Maggio R., Ruocco G., Gosti G. (2021). Asymmetric binomial statistics explains organelle partitioning variance in cancer cell proliferation, Communications Physics, 4.

The establishment of human motoneurons (MNs) from hiPSCs thanks to the combination of a short and efficient differentiation protocol and the stable insertion of the Hb9:GFP reporter, allowed the effective isolation of a large number of purified, hiPSC-derived MNs. Fluorescent-activated cell sorting (FACS) of differentiated human MNs was performed in gentle condition to reduce stress for the neurons. Isolated cells were used for whole-transcriptome analysis by RNA sequencing (RNA-seq) to identify both long and short RNA species significantly altered in MNs mutant of FUS, a RNA-binding protein linked to amyotrophic lateral sclerosis (ALS).

De Santis R., Santini L., Colantoni A., Peruzzi G., de Turris V., Alfano V., Bozzoni I., Rosa A. (2017). FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis, Stem Cell Reports, 9, 1450-1462. ​​​​​​

De Santis R., Alfano V., de Turris V., Colantoni A., Santini L., Garone M.G., Antonacci G., Peruzzi G., Sudria- Lopez E., Wyler E., Anink J.J., Aronica E., Landthaler M., Pasterkamp R.J., Bozzoni I., Rosa A. (2019). Mutant FUS and ELAVL4 (HuD) Aberrant Crosstalk in Amyotrophic Lateral Sclerosis, Cell Reports, 27, 3818-3831.e5. 

Garone M.G., Alfano V., Salvatori B., Braccia C., Peruzzi G., Colantoni A., Bozzoni I., Armirotti A., Rosa A. (2020). Proteomics analysis of FUS mutant human motoneurons reveals altered regulation of cytoskeleton and other ALS-linked proteins via 3′UTR binding, Scientific Reports, 10.  

Gentle FACS conditions were used to isolate mESCs derived from wild-type or knock out FUS mice expressing a GFP reporter under the control of the motoneuron-specific Hb9 promoter, differentiated into MNs. Total RNA of isolated mESCs-derived MN was sequenced to identify circular RNAs presence and expression levels to study their involvement in amyotrophic lateral sclerosis (ALS).

Errichelli L., Dini Modigliani S., Laneve P., Colantoni A., Legnini I., Capauto D., Rosa A., De Santis R., Scarfo R., Peruzzi G., Lu L., Caffarelli E., Shneider N.A., Morlando M., Bozzoni I. (2017). FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons, Nature Communications, 8. 

Capauto D., Colantoni A., Lu L., Santini T., Peruzzi G., Biscarini S., Morlando M., Shneider N.A., Caffarelli E., Laneve P., Bozzoni I. (2018). A Regulatory Circuitry Between Gria2, miR-409, and miR-495 Is Affected by ALS FUS Mutation in ESC-Derived Motor Neurons, Molecular Neurobiology, 55, 7635-7651. 

Biscarini S., Capauto D., Peruzzi G., Lu L., Colantoni A., Santini T., Shneider N.A., Caffarelli E., Laneve P., Bozzoni I. (2018). Characterization of the lncRNA transcriptome in mESC-derived motor neurons: Implications for FUS-ALS, Stem Cell Research, 27, 172-179 

Carvelli A., Setti A., Desideri F., Galfre S.G., Biscarini S., Santini T., Colantoni A., Peruzzi G., Marzi M.J., Capauto D., Di Angelantonio S., Ballarino M., Nicassio F., Laneve P., Bozzoni I. (2022). A multifunctional locus controls motor neuron differentiation through short and long noncoding RNAs, EMBO Journal, 41. 

In Alzheimer’s disease (AD) a pivotal role is played by two neurotoxic proteins that aggregate and accumulate in the central nervous system: amyloid beta and hyper-phosphorylated tau. Since the retina and the brain have been demonstrated to be associated to over a range of neurological diseases and respond similarly to neuropathological conditions, the idea of using the eye as a window through which monitor AD-related neurodegeneration process is growing. Using gentle FACS conditions, retinal tissues of triple-transgenic AD mouse model (3xTg-AD) was screened for the presence of pathological hallmarks during disease progression. The finding of inflammation, neurodegeneration and protein aggregation in the retina of these mice, leads to possible ocular biomarkers of AD disease.

Grimaldi A., Brighi C., Peruzzi G., Ragozzino D., Bonanni V., Limatola C., Ruocco G., Di Angelantonio S. (2018). Inflammation, neurodegeneration and protein aggregation in the retina as ocular biomarkers for Alzheimer's disease in the 3xTg-AD mouse model, Cell Death and Disease, 9. 

Natural killer (NK) cells are innate lymphocytes that represent the prototypical cytotoxic cells. Recently, other innate lymphoid cells (ILCs) with cytotoxic functions have been identified. Both type of cells requires the transcription factor STAT4 to elicit rapid effector responses and protect the body against pathogens. Here, a multiparametric flow cytometry approach was used to characterize NK and ILCs subsets in STAT4 deficient mice to investigate their role during intestinal inflammation.

Scarno G., Mazej J., Laffranchi M., Di Censo C., Mattiola I., Candelotti A.M., Pietropaolo G., Stabile H., Fionda C., Peruzzi G., Brooks S.R., Tsai W.L., Mikami Y., Bernardini G., Gismondi A., Sozzani S., Di Santo J.P., Vosshenrich C.A.J., Diefenbach A., Gadina M., Santoni A., Sciume G. (2023). Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation, Proceedings of the National Academy of Sciences of the United States of America, 120. 

A subset of NK cells endowed with low expression levels of both the activating receptors CD56 and CD16, named CD56lowCD16low NK cells, was characterized using a flow cytometry multiparametric approach. These cells have multifunctional properties and are more abundant in bone marrow (BM) than in peripheral blood (PB) of pediatric normal subjects. In this study, using FACS, NK cell subsets were isolated to investigate their development and functional recovery in a cohort of children undergoing haematopoietic stem cell transplantation (HSCT), a treatment for many malignant and non-malignant, hematological diseases.

Stabile H., Nisti P., Peruzzi G., Fionda C., Pagliara D., Pomonia B. L., Merli P., Locatelli F., Santoni A., Gismondi A. (2017). Reconstitution of multifunctional CD56lowCD16low natural killer cell subset in children with acute leukemia given α/β T cell-depleted HLA-haploidentical haematopoietic stem cell transplantation, OncoImmunology, 6