title: The VerIFAST: an integrated method for cell isolation and extracellular/intracellular staining.
abstract: Isolation and characterization of a specific subset of cells from a large heterogeneous population is necessary for studying rare subpopulations of cells. Existing methods require transfer or wash steps that risk causing loss of the rare cell population of interest. Integrated methods reduce loss, making these methods especially useful for reliable isolation of rare cell populations. In this report, we demonstrate the VerIFAST, a device that builds upon the simplified workflow of the Immiscible Filtration Assisted by Surface Tension (IFAST) to integrate a method for cellular isolation with methods for extra- and intracellular staining. First, a front-end purification step allows cells and unwanted particulates to passively settle out of the operational path of the paramagnetic particles, resulting in good efficiency of capture (>80%) and purity (>70%) with a single virtual wall traverse. Second, a Sieve Chamber is used downstream of the isolation chamber that removes excess unbound paramagnetic particles (PMPs) and performs complex multi-step washing procedures without centrifugation or transfer steps. Further, cellular staining can be performed in the device and is demonstrated for extracellular epithelial cell adhesion molecule (EpCAM), intracellular pan-cytokeratins, and Ki-67.
authors.affiliation: Department of Biomedical Engineering, Wisconsin Institutes for Medical Research, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Casavant
authors.forename: David J
authors.initials: DJ
authors.lastname: Guckenberger
authors.forename: Scott M
authors.initials: SM
authors.lastname: Berry
authors.forename: Jacob T
authors.initials: JT
authors.lastname: Tokar
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Lang
authors.forename: David J
authors.initials: DJ
authors.lastname: Beebe
mesh_terms: D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D000071858:Epithelial Cell Adhesion Molecule; D004867:Equipment Design; D005374:Filtration / Q000295:instrumentation* / Q000379:methods; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D013500:Surface Tension
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule