1. FUCCI Transduction and Cell Culture

1. FUCCI transduction
   1. Create cell lines stably expressing the FUCCI constructs mKO2-hCdt1 (30-120) and mAG-hGem (1-100) ¹⁵ using lentivirus co-transduction as previously described ⁷.
      Note: The FUCCI system is now commercially available.
   2. Generate sub-clones with bright fluorescence by single-cell sorting. Sort single cells positive for both AG and KO (yellow) by fluorescence activated cell sorting into a 96-well plate as previously described ^7,16.
2. Melanoma cell culture
   1. Culture C8161 human melanoma cells as previously described ^7,17.

2. 3D Spheroid Formation (as previously described ^3,9)

1. Agarose plate preparation
   1. Dissolve 1.5% agarose (or noble agar) in 100 ml of Hank’s balanced salt solution (HBSS) or phosphate buffered saline (PBS) by boiling in the microwave for 3-5 min with swirling. Make sure the agarose is fully dissolved.
   2. Immediately dispense 100 μl of the agarose solution per well to a flat-bottomed 96-well tissue culture plate using a multi-channel pipette.
      Note: Agarose may solidify in the tips after a short period of time, to overcome this problem tips must be changed between plates if making more than one agarose plate.
   3. Leave 96-well plate on a flat surface to harden agarose for at least 1 hr.
2. Cell preparation and overlay onto agarose
   1. Grow cells to approximately 80% confluency in a T75 flask. Wash with 10 ml of HBSS.
   2. Detach cells using 1.5 ml of 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA) and resuspend in 10 ml of normal medium.
   3. Count cells using a hemocytometer ¹⁸ or other automated counting method.
   4. Resuspend cells to a final concentration of 25,000 cells/ml in normal medium.
   5. Overlay agarose wells with 200 μl of the cell suspension for a final number of 5,000 cells per well.
   6. Return plates to the cell culture incubator (5% CO₂ and 37 ˚C) to form spheroids over approximately 3 days.
      Note: The time taken to form a spheroid varies between different cell lines. Some cell lines do not form spheroids using this method at all.
   7. Image spheroid morphology with an inverted microscope using a 4X objective and a phase contrast filter. A cell line that successfully forms spheroids will produce compact, roughly spherical cell aggregates, and there should be only one spheroid per well.
      Note: Optional: Once spheroids have formed, they can be transplanted into collagen or other matrix for further analysis of growth and invasion ^3,7,17. To remove spheroids from collagen, treat with 500 μl of 2 mg/ml collagenase at 37 °C until the collagen is dissolved enough for the spheroids to come free into the media (approximately 30 min). Gently remove spheroids using a 1 ml pipette.
   8. For sectioning/imaging fix spheroids (either isolated from collagen or grown for 3-5 days on agarose) in 10% neutral buffered formalin (CAUTION: Formalin should be used in a fume cupboard) for at least 2 hr at room temperature (RT), or overnight (O/N) at 4 °C. Spheroids may be stored in HBSS at 4 °C for several days.

3. Spheroid Vibratome Sectioning

1. Dissolve 5 g of low melting point agarose in 100 ml of HBSS in a 500 ml glass bottle in a microwave. Use a medium heat setting with swirling. CAUTION: Keep the lid loose when boiling the solution so that the pressure can be released. Use protective heatproof gloves to protect hands from burns when handling the hot glass bottle.
2. Wait until the agarose cools slightly – so the bottle can be touched without burning hands .
3. Pour approximately 2 ml of agarose into the bottom of a Coulter counter cup or similar plastic mold. Immediately aspirate a fixed spheroid (see section 2.2.8) using a 1 ml pipette in the smallest volume of liquid possible. Add spheroid to the agarose. A few spheroids (up to 10) may be added per cup.
4. Cover the spheroids with 2 ml more of agarose. Do this quickly to avoid the agarose hardening before the second layer is added.
5. Allow the agarose to harden at RT in the dark for at least 3 hr. At this point cups may be stored for a short time at 4 °C with 2-3 ml of HBSS overlaid to prevent dehydration.
6. Remove agarose from the cup. Trim the agarose containing the spheroids so that it fits onto the vibratome metal block, and spheroids are close to the top of the agarose. Spheroids can be seen as small white objects inside the agarose. Super glue the agarose to the block.  
7. Fill the vibratome with distilled water and mount the block in the vibratome. Set the vibratome to cut 100 µm sections using low speed (setting 2) and high vibration (setting 8). Set the cutting blade angle to 16°. Turn vibratome on, turn on the vibratome light and cut sections from the top of the agarose until 100 µm spheroid sections are cut. Place cut spheroid sections into HBSS in a 24-well plate.
8. Choose middle sections for image analysis. Mount sections on slides by transferring the section flat onto the slide using tweezers. Fill a 10 ml syringe barrel with vacuum grease; add a thin line of vacuum grease around the edges of the section in order to prevent the mounting media from running off the edges of the slide and help seal the section under the coverslip. Cover the section with mounting medium and a coverslip. Seal edges of coverslip with nail polish. Store slides at 4 °C before imaging.

4. Confocal Image Acquisition

1. Take a z-slice image of a middle FUCCI spheroid section using a 10X or 20X objective as previously described ⁷. Make sure no overlap or crosstalk occurs between the Kusabira Orange2 and Azami Green. If comparing image analysis between multiple spheroid sections, keep laser power and other settings the same between samples.

5. Hoechst Dye Diffusion Assay for Flow Sorting

1. Transfer live spheroids (3-5 days after agarose seeding) to a 15 ml tube. Let spheroids gravity settle to the bottom of the tube. Several spheroids may be stained per tube. Approximately 20 spheroids are needed to gain sufficient cell numbers for flow cytometry.
2. Remove excess medium and add 1 ml of 10 μM Hoechst diluted in normal medium. Incubate spheroids at 37 °C for approximately 1 hr. Use gentle flicking to resuspend the spheroids once or twice during incubation.
   Note: The incubation time may be varied to alter how far the Hoechst dye penetrates into the spheroids. This will vary based on the spheroid size, density and cell line. For some large/dense spheroids there may be a maximum dye penetration that is less than 100%.
3. Wash the spheroids gently with 5 ml of HBSS using gravity settling.
   1. Optional: For imaging of dye penetration: Fix spheroids with 10% neutral buffered formalin for at least 2 hr at RT or O/N at 4 °C. Prepare vibratome sections, mount on slides and image on the confocal as described in steps 3 and 4 above.
4. To prepare cells for flow cytometry, trypsinize spheroids with 1 ml of 0.05% trypsin/EDTA at 37 °C for approximately 30 min with flicking every 10 min to break the spheroids apart.
   Note: Spheroids are difficult to get into single cell suspension, conditions may need to be optimized. Viability of C8161 4 day old spheroids after this process is approximately 95%.
5. Stain cells with a near-infrared live/dead stain according to the manufacturers protocol. Wash with HBSS then fix with 1 ml of 10% neutral buffered formalin for approximately 30 min. Cells may be stored in HBSS at 4 °C for several days before flow analysis.

6. Flow Cytometry Analysis of Hoechst Stained FUCCI Spheroids

1. Ensure cells are not clumped together by passing them through a cell strainer. Resuspend the Hoechst stained FUCCI spheroids in ice cold FACS wash (PBS with 5% Fetal Calf Serum) at a concentration of 1-5 x 10⁶ cells/ml. Transfer 200 µl of sample to 5 ml round bottom tubes.
2. Prepare the following single colour control samples as described in 6.1: un-stained melanoma cells; adherent melanoma cells stained with 10 µM Hoechst for 1 hr; Azami Green only expressing melanoma cells; and Kusabira Orange only expressing melanoma cells.
   Note: Single colour controls may be fixed in formalin and stored in FACS wash with 0.1% Sodium Azide at 4 °C for weeks or even months.
3. Run single cell suspensions on a flow cytometry analyzer with appropriate lasers and single colour/unstained controls as previously described ^7,16.
4. Use commercial cytometry software such as FlowJo to analyze the inner vs. outer spheroid cells as follows:
   1. Remove debris by gating on the main cell population in Forward Scattered Light (FSC) vs. Side Scattered Light (SSC).
   2. Remove doublets by gating on single cells using FSC (Area) vs. FSC (Height)
   3. Gate for live cells by gating on the live/dead low population.
   4. Define Hoechst high cells, gated based on the positive signal from the adherent cells stained with Hoechst, as “outer” cells.
   5. Define Hoechst low or negative cells, gated based on the signal from the un-stained control, as “inner” cells.
   6. After gating for the inner and outer populations, cells may be further gated for FUCCI red (G1), yellow (early S), green (S/G2/M) or negative (early G1).
      Note: Compensation using the single colour controls may be required to properly define the FUCCI red, yellow, green and negative populations.
5. Once the assay has been optimized and Hoechst penetration validated via confocal microscopy, run cells without fixing on a flow cell sorter as previously described ^7,16 for further analysis of live cell sub-populations.

7. Image Analysis of FUCCI Spheroid Sections

1. Open software e.g. Volocity, choose the Quantitation ONLY configuration.
2. Create a new library. Import the FUCCI spheroid confocal image file into the software by dragging the raw data file from the confocal into the library. Alternatively, use the File >Import command.
3. Go to the measurements tab to build an image analysis protocol. The Protocol is built by dragging and dropping the appropriate commands in the correct order as follows in the protocol window.
4. Find objects in the green channel: Drag the find objects command (found in ‘Finding’) into the protocol window, select the correct channel in the find objects protocol.Add an open command (found in ‘Processing’), then a separate touching objects command (found in ‘Processing’ – this will separate cells). Exclude objects by size <50 μm (found in ‘Filtering’ – this will exclude small non-cellular objects).
   Note: Variables such as the find objects threshold, number of open iterations, the size of objects to be separated and the size of objects to exclude must be manually optimized until the object masks match the image.
5. Find objects in the red channel: Repeat find objects as above for the red channel. Green and red protocols may need to be adjusted separately.
   Note: Due to nuclei being in G2, green nuclei are often slightly larger.
6. Confirm objects found are accurate: Confirm red and green objects match the red and green nuclei in the image by turning off and on the green and red channels (using the hide or show channel command) while displaying the red and green masks found by the protocol. Feedback options (Measurements >Feedback options) may be used to modify the appearance of the object masks.
7. Find yellow objects (early S phase cells): To find yellow cells, add an intersect red with green cells command (found in ‘Combining’). Exclude objects by size (<50 μm, found in ‘Filtering’) to exclude any small non-cellular objects.
8. Find exclusively red objects (G1 phase cells): To find exclusively red nuclei, subtract the yellow cells from the red cells (found in ‘Combining’). Exclude objects by size <50 μm, (found in ‘Filtering’) to remove any small non-cellular objects created.
9. Find exclusively green objects (S/G2/M phase cells): Repeat for the green channel to find exclusively green.
   Note: If there are still non-cellular objects remaining, a filter population command may be added to the Exclusive green or Exclusive red protocols (found in ‘Filtering’) to retain only objects with a shape factor greater than 0.25. This will remove non-circular objects.
10. Find the spheroid outline: Find the spheroid outline using a find objects command (found in ‘Finding’) with the green or red channel (whichever has more cells around the edge of the spheroid). A much lower threshold (found in the find objects variables) will need to be used to find the spheroid outline compared to individual cells.
    1. Use a close command to join objects (found in ‘Processing’), then fill holes in objects (found in ‘Processing’), then use a fine filter (found in ‘Filtering’) to remove noise from objects. Finally, exclude objects by size to remove smaller objects <30,000 μm (depending on the spheroid size).
       Note: This protocol will need to be optimized manually till the spheroid outline is accurate. A brightfield image may also be used – however this is usually less accurate with a spheroid section, and an invert command (found in ‘Combining’) will need to be used.
11. Measure distances of nuclei to spheroid outline: Measure distances (found in ‘Relating’) from the yellow, exclusively green and exclusively red populations from the cell centroid to the edge of the spheroid outline.To visualize the minimum distances, which should be from the cell to the nearest spheroid edge, turn on show distances in the relationships tab in feedback options (Measurements >Feedback options >Relationships).
12. Save the protocol. This protocol may be re-applied to other images using the Measurements >Restore protocol command.
13. Create a measurements item (Measurements >Make Measurement Item). Data may be analyzed using the analysis functions.
    1. Go to the analysis tab in the measurements item. Go to the Analysis menu (Analysis >Analyze) and analyze the minimum distance from the cell centroid (red, green or yellow) to the spheroid edge, summarized by count and organized by population.
    2. To count the number of cells found at a certain distance from the edge create a filter (Analysis >Filter). The filter for minimum distance in Figure 2 is based on Hoechst penetration (e.g. minimum distance is less than 80 gives the “outer” population). Alternatively, export raw data to a spreadsheet for further analysis.