Fv100 confocal laser scanning microscope

MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PMA₄/PAAm₄ coated microfluidic slides and PMA₄/PAAm₄ coated microfluidic slides with DOTAP (50,000 cells/channel). Cells were incubated for 1 hr to allow for tethering. After 1 hr, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to top port on the DOTAP slides. This wash was to ensure only tethered cells were analyzed. After this wash, CellMask orange cell membrane dye was added to each channel to a final concentration of 1:10,000. Cells were treated with 5μM colchicine for 15 mins and 1μg/ml paclitaxel for 120 mins. McTN imaging was done on an Olympus FV100 confocal laser scanning microscope at 60x magnification. Five 1μm slices and 20 frames at a 10 sec frame rate were taken for at least five image sets for each condition. The number of McTNs on each cell was manually counted on five cells per condition using the maximum intensity z-projection at the last frame.

Human UCB‐derived Lin⁻/CD133⁺/CD45⁻ cells (VSELs), and corresponding Lin⁻/CD133⁺/CD45⁺ cells (enriched for HSPCs), and MSCs were plated, fixed in 3.7% paraformaldehyde for 15 min. at 4°C, and then permeabilized with 0.1% Triton X‐100 for 5 min. After blocking with 2.5% BSA, the cells were subjected to immunostaining with the following primary antibodies: follicle‐stimulating hormone receptor (FSHR, 1:200, rabbit polyclonal antibody; Santa Cruz Biotech), luteinizing hormone/choriogonadotropin receptor (LHR, 1:200, rabbit polyclonal antibody; Santa Cruz Biotech), androgen receptor (1:50, rabbit polyclonal antibody; Neomarkers; Fremont, CA, USA), and oestrogen receptor alpha (1:500, mouse monoclonal IgG antibody; ThermoScientific; Grand Island, NY, USA). These antibodies were diluted in 2.5% BSA and incubated with the cells for 1 hr 15 min. at 37°C. Appropriate Alexa Fluor 594 Goat Anti‐Rabbit IgG and Alexa Fluor 488 Goat Anti‐Mouse IgG were used as secondary antibodies (1:400, all from Invitrogen, Life technology, Carlsbad, CA, USA), and incubated with the cells for staining for 75 min. at 37°C. In control experiments, cells were stained with secondary antibodies only. In all experiments, the nuclei were labelled with DAPI, and the fluorescence images were collected with a FV100 confocal laser‐scanning microscope (Olympus America Inc., Center Valley, PA, USA).