488 conjugated wheat germ agglutinin

The channels in MS-chip and Tygon tube connected to the chip inlet were wetted with PBS and then kept with 0.5% BSA in PBS for 1 hour. BSA blocks the surface and further prevents nonspecific adhesion of cells to PDMS. Membranes of control and hENT1 knockdown cells were stained using Alexa Fluor 594 or 488-conjugated wheat germ agglutinin (Invitrogen, Grand Island, NY), respectively. The cell mixture in equal amounts at a final density of 1×10⁵ cells/ml was prepared. Suspended cells were then applied to the MS-chip via a Tygon tube. During the experiment, compressed nitrogen gas was applied to the cell suspension at a pressure of ∼10 psi (69×10³ Pa). A typical separation lasted ∼15 min, and average flow rate was controlled at 1–2 mL/h. Cells applied to the MS-chip were imaged by fluorescence microscopy (IX81, Olympus). Number of cells retained on chip after separation was counted by Image J.

Hearts were isolated and fixed in 4% (vol/vol) paraformaldehyde in PBS for 48 hr at 4°C with gentle shaking. Hearts were dehydrated, embedded in paraffin, and sectioned. Heart sections were stained with hematoxylin and eosin (H and E) and Picrosirius red using standard procedures. Fibrosis was quantified using Pircorsirius red staining and ImageJ software (NIH, Rockville, MD). For immunofluorescent staining, tissue sections were deparaffinized and subjected to antigen retrieval with Citra buffer (BioGenex, Fremont, CA). Tissue sections were incubated with 488-conjugated Wheat Germ Agglutinin (Invitrogen, Carlsbad, CA) to label the cell membranes and cardiomyocytes were immunostained using a primary antibody for cardiac troponin-T (Abcam, Cambridge, MA) and an Alexa Fluor 555 secondary antibody (Invitrogen, Carlsbad, CA). Coverslips were mounted using VECTASHIELD Antifade Mounting Media with DAPI (Vector Laboratories, Burlingame, CA) and confocal images were taken of the mid-LV free wall with a Zeiss LSM-800 using a 40X oil objective. Cardiomyocyte cross-sectional area was assessed using Fiji Software. Specific catalogue numbers for the reagents used for immunohistochemistry can be found in the Key Resource Table.