Anti-CD9 monoclonal antibody, clone Bu16

The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

The distal tubule (DT) helps regulate blood pressure and electrolytes. We describe a novel, autosomal recessive, morphofunctional DT abnormality in inbred mice evident as columnar alternations and age related cystic changes. This abnormality developed in both sexes of DBA/2Cr. Similar phenotypes were observed in A/J, C3H/He, DBA/1J, and FVB/N strains, but not in AKR/N, BALB/c, or C57BL/6N strains. In DBA/2Cr, abnormal DT localized to straight and convoluted segments and showed IL-36 alpha DT injury marker expression. However, DT epithelial proliferation, examined by bromodeoxyuridine incorporation, was not remarkably altered with the progression of abnormality. Abnormal DT epithelial cells in DBA/2Cr displayed elongated primary cilia, loose intercellular adhesions, and numerous vesicles with altered localization of CD9, Na+/K+ ATPase, and E-cadherin, indicating altered cell function, adhesion, and polarity. DBA/2Cr-type D12Mit182-012Mit83 was identified as a candidate locus designated DBA/2 renal cyst (drecy). Within drecy, the gene regulated by estrogen in breast cancer protein (Greb1) transcript variant 2 was significantly up-regulated in DBA/2Cr kidney versus C57BL/6N. Greb1 localized to DT cytoplasm in C57BL/6 and to cytoplasm and nucleus in DBA/2Cr. Grebl-overexpressing M-1 kidney cells showed an altered epithelial-mesenchyme phenotype. B6.D2-(D12Mit182-D12Mit83) congenic mice carrying drecy did not show DT abnormalities, whereas DBA/2Cr x B6.02-(012Mit182-012Mit83) mice did. Identification of this novel DT abnormality regulated by a DBA/2Cr mouse chromosome 12 derived locus and additional genetic factors improve the understanding of DT pathogenesis.