Data dissemination: Reference Manual Segmentations

A-10 and NIH-3T3 cells (5.5MB)
A-10 rat smooth muscle cells and NIH-3T3 mouse fibro- blasts (ATCC, Manassas, VA) were maintained in Dulbecco’s Modified Eagles Medium (DMEM/10 %FBS, Mediatech, Herndon, VA) supplemented with glutamine, nonessential amino acids and occasionally penicillin/streptomycin (Invi- trogen, Carlsbad CA) in 5% CO2 at 37 degree C. For the experiment, the cell lines were seeded at 800 and 1,200 cells/cm2, respec- tively, in three-wells of a six-well tissue culture treated polysty- rene plate (353046, BD Falcon, Franklin Lakes, NJ) in mainte- nance media, and placed in the incubator for about 20 h. The media was removed; the cells were rinsed with PBS and fixed for 3 h with 1% (v/v) formaldehyde in PBS at 258C. The cells were stained with PBS containing 0.02% (v/v) TritonX-100 (Sigma, St. Louis, MO), 0.5 lg/mL TxRed c2 maleimide (Invitrogen) (5 mg/mL in DMSO stock), 1.5 lg/mL DAPI) (Sigma) (1 mg/mL in DMSO stock) for 4 h, rinsed with PBS, PBS containing 1% BSA and PBS, sequentially. Fixed and stained cells were covered with PBS, stored at 4 degree C, and imaged within two days. side views of this 3D volume.
Live NIH 3T3 cells (89MB)
NIH 3T3 fibroblast cells: Prior to an acquisition, cells were seeded into a 6-well plate at 1200 cells/cm2. Images were acquired every 15 min for greater than 62 h using a 10X (0.3 NA) objective on Zeiss 200M inverted microscope equipped with an automated stage, a collimated blue LED (470 nm) fluorescence excitation source and a CoolSNAP HQ CCD camera. A 0.63x demagnifying lens was positioned in front of the CCD. Cells were maintained throughout the experiment in a humidified 5 % CO2 balanced-air atmosphere at 37 °C using a microscope incubation chamber. Before the start of each experiment, 12 stage locations were manually identified and the x, y, and z positions were stored within the acquisition software. Immediately before the time lapse acquisition started, each field was exposed to the fluorescence excitation for 30 sec to reduce background fluorescence from the growth media. At each position the image acquisition software controlled the following operations sequentially:

  1. the transmitted light shutter was opened
  2. auto-focusing was performed on the phase contrast image
  3. a phase contrast image was acquired
  4. the transmitted light shutter was closed
  5. the fluorescence illumination shutter was opened
  6. a GFP image was acquired
  7. the fluorescence illumination shutter was closed

GFP fluorescence was collected with a standard GFP filter cube set in the optical train. The CCD acquired images using 2x2 binning for 0.05 s (for phase contrast) and 0.3 s (for GFP fluorescence). All live cell data were acquired in the presence of phenol red free media. Three time-lapse image sets were generated, each containing 3 well-to-well replicates with roughly 238 time-lapse images at 520x696 pixels each.