Low melt agarose

Trypsinized cells were resuspended in RPMI-1640 media without phenol red (#R8755, Sigma–Aldrich) containing 0.35% low-melt agarose (#1613111, Bio-Rad) and 10% FBS (Sigma). The cell suspension was plated on top of a 0.5% agarose base layer. The cells were allowed to grow in a Forma Steri-Cycle CO₂ incubator (Thermo Fisher) CO₂ incubator at 37°C with 5% CO₂ for 3 weeks and stained with 0.05% crystal violet (Gentian Violet, ICM Pharma, Singapore). Images of colonies were acquired using a dissecting microscope (SZX-12, Olympus, Tokyo, Japan) and the colony numbers and sizes were quantitated using Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij/, 1997–2016).

At the appropriate time point, larvae were stained with a 0.2% calcein (Sigma) solution in fish water. A subset of fish were also stained with a 0.05% alizarin red (Sigma) solution in fish water 3 days prior to calcein staining. For imaging, stained zebrafish larvae were anesthetized in 0.01% MS-222 (Sigma) and mounted into borosilicate glass capillaries using 0.75% low melt-agarose (Bio-Rad) diluted in system water containing 0.01% MS-222. Capillaries were set on a custom 3-D printed holder to aid manipulation and rapid orientation of the specimen. Three-channel (GFP, DsRED, DAPI) images were collected on a high-content fluorescent microscopy system (Zeiss Axio Imager M2, constant exposure settings for all experiments) using a 2.5x objective (EC Plan-Neofluar 2.5x/0075). For each fish, a composite image stack (usually 3/1 images in the x/y directions; and optimized to 30-70 μm slice intervals in the z direction across the entire region of interest, usually about 9 slices; all at 2.58 μm/pixel) was acquired in mediolateral and anteroposterior views. Maximum intensity projections were generated from image stacks in Fiji for analysis. Following imaging, fish were collected for genomic DNA extraction and genotyping.

The assays were performed in 6-well plates using the low-melt agarose (Bio-Rad, Hercules, CA). The bottom layer was 0.8% agar diluted in DMEM with 20% FBS. For the top layer, cells were first suspended in 0.48% agar in DMEM with 20% FBS and then plated on top of the bottom layer. Additional 1 ml of DMEM was added to each well. Cells were incubated in humidified 5% CO₂ incubator at 37 °C for 2–3 weeks.

Samples were prepared as previously described (Selmecki et al., 2005 (link)). Cells were suspended in 300 µL 1.5% low-melt agarose (Bio-Rad) and digested with 1.2 mg Zymolyase (US Biological). Chromosomes were separated on a 1% Megabase agarose gel (Bio-Rad) in 0.5X TBE using a CHEF DRIII apparatus. Run conditions as follows: 60 s to 120 s switch, 6 V/cm, 120° angle for 36 hrs followed by 120 s to 300 s switch, 4.5 V/cm, 120° angle for 12 hrs.

Embryos at the 10–13 somite stage were manually dechorionated, embedded in 1% low-melt agarose (Bio-Rad) in a glass bottom dish with thickness of No. 1.5 (MatTek Corporation). The dorsal side of the embryo faces the cover glass. Experiments were performed at a room temperature (22°C).

A Zeiss Laser Scanning Microscope-780, 880, or Olympus FluoView1000 laser scanning microscope was used to generate the fluorescent images. Settings were optimized to visualize GFP, YFP (laser wavelength, 488 nm; detection wavelength, 493 to 598 nm) or chlorophyll (laser wavelength, 545 nm; detection wavelength, 604 to 694 nm). All direct comparisons of images were performed with the same settings. Maximum intensity projections were generated from z-stacks of multiple tiles to visualize the entire apical region of the gynoecium/silique. Samples were mounted on a glass slide with 1% low-melt agarose (Bio-Rad) and submerged in de-ionized water or mounted in 30% glycerol and a cover slip was placed on top and sealed with nail varnish.