Hcs cellmask red

NS-1 cells were fixed with 4% paraformaldehyde after treatment, permeabilized with 0.2% Triton X-100 for 15 min, washed with PBS, and then stained with HCS CellMask Red (0.5ug/ml, Invitrogen) for 30 min at room temperature. Images were taken using a fluorescent microscope (DM6000; Leica Microsystems, Buffalo Grove, IL, USA). For measurement of neurite length, images were randomly selected and taken using a fluorescent microscope (DM6000; Leica Microsystems). The length of neurites of 150–200 single cells per condition was analyzed with LAS AF software (Leica Microsystems).

3 × 10⁴ cells were seeded on coverslips and stained with HCS CellMask Red (Invitrogen) and DAPI. Numerous fields were imaged on DeltaVision Image Restoration Microscope to capture at least 100 cells per population. Cell size parameters were measured using Cell Profiler 2.0. Debris was filtered out by generating a histogram of (cytoplasmic area—nuclear area) and applying a minimum threshold in R. To calculate coefficient of variation (s.d./mean) for each clonal population, a sampling size was determined from the clonal population with the fewest cells analysed (96 cells in the MDA-MB-231 population and 206 cells in the CN34 population). Subpopulations with more cells imaged than the sampling size were sampled 100 times, with the average CV used in the final analysis. For each subpopulation, coefficient of variation was calculated for each size parameter, which includes cell area, cytoplasmic area, nucleus, perimeter, major axis length, and minor axis length. Principal component analysis was performed using all subpopulations on the coefficient of variations for all morphological parameters. The first principal component accounted for 93 and 91% of the variance in MDA and CN34 cell lines, respectively, and is used for all size variation analyses.

Reagents were sourced from the following vendors: NS-1 (Cellomics, Inc., Pittsburgh, PA), PC12 (ATCC, Manassas, VA), 2.5S murine NGF (Bachem Inc., Torrance, CA), RNA extraction kit and Turbo DNA-free kit and RETROscript Kit (Ambion Inc., Carlsbad, CA), RT PCR primers (Eurofins MWG Operon, Huntsville, AL); RT² SYBR Green ROX qPCR Mastermix (QIAGEN, Valencia, CA), MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO), DMEM and RPMI 1640, HCS CellMask Red™, and Alexa Fluor 488 Donkey anti-rabbit antibody (Invitrogen, Carlsbad, CA), fetal bovine serum (FBS) (Hyclone, Logan, UT), heat inactivated horse serum (Lonza, Walkersville, MD), and L-glutamine and 1% Penicillin/Streptomycin (BioWhittaker, Walkersville, MD), Mouse anti-ChAT and Rabbit anti-M2 antibody (EMD Millipore, Billerica, MA), Goat anti-VAChT (Promega, Madison, WI), Rabbit anti-TrkA (Cell Signaling, Tech., Danvers, MA), and Super Signal Pico Chemiluminescent Substrate (Thermo Fisher Sci., Grand Island, NY).