In general, however, minor differences between NPS and FCM could mainly be attributed to their different gating strategies, with NPSs size gating resulting in the potential inclusion of cells other than blasts. Conclusions NPS is a simple, yet accurate method to screen cells for multiple surface markers simultaneously. of comparable sizeAP-1060 and NALM-1based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside. Flow cytometry (FCM) is one of the cornerstones of biomedical research and clinical diagnostics. With its ability to screen individual cells for multiple protein epitopes simultaneously and subsequently identify subpopulations of cells, FCM has had a profound impact in a broad range of areas including immunology,1?3 cancer,4,5 and regenerative medicine.6,7 Recent advances in both fluorochrome and laser technologies have dramatically increased the number of proteins that can be screened simultaneouslyfrom 2 to the current state-of-the-art of 208,9further advancing these fields. Despite this tremendous increased capability, multi-color FCM can be difficult to implement given that spectral emission overlap significantly increases with the number of fluorochromes utilized simultaneously, and highly complex analysis is necessary to decouple such overlap.3,9 Additional challenges include the following: the high cost per assay to the user, lengthy sample preparation steps, and multiple control tests that need to be performed separately. Furthermore, because of its overall complexity, the need for frequent calibration, and high cost as an instrument, multicolor FCM is often located in a central facility and operated by a skilled AEG 3482 technician. Most recently, mass cytometry, or CyTOF, which combines FCM with mass spectrometry and can screen more than 70 parameters simultaneously, has been introduced.3,9 Although it is a paradigm-shifting technique, CyTOF does have one distinct disadvantage: cells are vaporized and are therefore not available for collection for secondary analysis or culture. Numerous lab-on-a-chip technologies for cell screening have also been introduced. Examples include the true miniaturization of fluorescence-activated cell sorting10?12 and dielectrophoretic or impedance cell characterization.13?17 Although successful in targeted applications, these on-chip technologies have a number of distinct disadvantages, ranging from the need for exogenous labeling with fluorophores or magnetic beads, to the limited parameters that can be screened because the hardware is not yet as sophisticated as AEG 3482 that in FCM, AEG 3482 to the inability to distinguish cellular subpopulations with similar morphologies or physical properties (e.g., dielectric constants, cell size, etc.). Here, we describe a unique label-free, microfluidic method that employs Node-Pore Sensing (NPS)18 to screen single cells for both size and multiple cell-surface epitopes, simultaneously. NPS is based on measuring the current pulse caused by a cell transiting a microchannel that has been segmented by a series of inserted nodes (Physique ?(Figure1).1). Like resistive-pulse sensing (RPS),19?22 i.e., the Coulter-counter theory,23 the magnitude of the current pulse corresponds to cell size; however, unlike RPS, the current pulse in NPS is usually modulated, reflecting both the number and spacing of the nodes in the channel.18 When the individual segments between the nodes are functionalized with different antibodies corresponding to distinct cell-surface antigens, cells whose antigens can interact specifically with the functionalized antibodies in a particular segment will travel more slowly through that section of the channel than through the isotype-control segment. Surface-marker identification, and ultimately phenotypic profiling, is usually thus accomplished by comparing transit times within the modulated pulse. Unlabeled AEG 3482 Fgf2 cells remain viable and are available for downstream analysis and/or culturing post screening. We demonstrate the versatility of NPS by successfully screening cells from established human cell lines for their specific phenotypic profiles and by distinguishing cell types in a mixed population based on surface-marker profiles. Moreover, we demonstrate the potential clinical value of NPS by immunophenotyping primary human bone marrow samples from acute myeloid leukemia (AML) patients. Overall, we.