Maintenance of SKOV-3 cells before spheroid generation

After thawing from liquid nitrogen, cells were maintained in Nunclon Delta T25 cell culture flasks in Gibco McCoy’s 5A medium supplemented with 10% Gibco  FBS and 1% Pen-Strep for 1 passage before seeding for spheroid generation. ATCC protocol was followed for subculturing.

Materials required

Protocol for spheroid generation

  1. On the day of the experiment, medium from the flask was aspirated, the cells were washed once in 1X PBS and dissociated using 1–1.5 ml of TrypLE reagent.
  2. The TrypLE reagent was neutralized using 4 volumes of complete medium, and live-cell count and viability were captured using Countess II cell counting chamber. Cells with >90% viability were taken for spheroid generation.
  3. The stock of cells was diluted 1:10 to 1:20 in complete medium to make calculations for cell seeding density easier.
  4. Seeding cell number was calculated using the cell seeding calculator.
  5. Required number of cells were seeded in respective wells of the Nunclon Sphera plate using a multi-channel pipette. The final volume was maintained at 100 μl.
  6. The plate was centrifuged at 250 g for 5 minutes and placed in the incubator. This is Day 0.
  7. Spheroids were ready on Day 1 (18–24 hours).

Skip to Cell viability LIVE/DEAD visualization protocol

Cell Seeding Calculator

Number of live cells/mL
(as determined by the Countess Automated Cell Counter)

Number of cells to seed per well
(user-specified number)

... µL

Volume of cell suspension that contains
the specified number of cells


  • Fill the outermost wells of the plate with PBS to prevent evaporation of media during incubation.
  • Make sure to have a single-cell suspension before seeding cells. Clumps will not generate uniform spheroids.
  • We have observed that centrifuging the plate after seeding cells facilitates cell aggregation leading to uniform spheroid formation consistently. However, this step is optional.


  • There is significant compaction of the cell aggregate on Day 1 compared to Day 0.
  • Compared to Day 1, there is increased compaction as well as increased cell death on Day 3.
  • Beyond Day 3, the compactness of the spheroids is compromised.
  • Cell seeding densities of <5,000 and >10,000 do not form good spheroids.

Morphology of SKOV-3 spheroids

Microscopic image of multiple SKOV-3 spheroids growing in culture and at different seeding densities

2,500–20,000 SKOV-3 cells were seeded for spheroid generation and brightfield images of spheroids at Day 0, Day 1 and Day 3 were captured using EVOS M7000 microscope under 4x magnification. Scale bar denotes 650 μm.

Characterization of SKOV-3 spheroids

Visualizing live and dead cells using LIVE/DEAD Viability/Cytotoxicity kit

  1. The populations of live and dead cells in Day 1 and Day 3 SKOV-3 spheroids were visualized by staining them with the reagents in the LIVE/DEAD Viability/Cytotoxicity kit.
  2. Working solution was prepared by adding Calcein AM and EthD-1 at a final concentration of 2 μM each in 1X PBS. (See our application note for more information on fluorescence staining of spheroids).
  3. NucBlue Live ReadyProbes Reagent was added (2 drops per ml) to the working solution for nuclear staining of the spheroids. 100 μl of this working solution was then added to each well of the 96-well plate containing 100 μl of spent medium. The plate was then incubated at 37°C for 3 hours.
  4. Following this, the plate was centrifuged at 250g for 5 minutes at room temperature, the medium was exchanged 1:1 with PBS and centrifuged again to minimize background during fluorescence imaging.
  5. Fluorescence images were captured using CellInsight CX7 high-content screening platform under 4X objective. Each image is a maximum intensity projection of 10 μm z-stacks (15–25 stacks depending on spheroid size).
Note: With increasing spheroid diameter, there is reduced penetration of dyes and an increase in the dead cell population (red staining).
microscopic views of fluorescently stained SKOV-3 spheroids on Days 1 and 3 at various seeding densities
Representative images of Day 1 (top panel) and Day 3 (bottom panel) SKOV-2 spheroids showing the live (green fluorescence) and dead (red fluorescence) cell population. Scale bar = 300 μm. The nucleus has been pseudocolored for better visibility.