Maintenance of T47D cells before spheroid generation

After thawing from liquid nitrogen, cells were maintained in Nunclon Delta T25 cell culture flasks in Gibco RPMI 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


  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 200 μl.
  6. The plate was centrifuged at 250 g for 5 minutes and placed in the incubator. This is Day 0.
  7. Media was changed 1:1 every alternate day. (aspirate 100 μl of spent medium and add 100 μl of fresh medium). The plate was centrifuged at 250 g for 5 minutes after media change and placed back in the incubator.
  8. Spheroids were ready on Day 4–7 depending on seeding number.

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.
  • For T47D cells, seeding density of >8,000 cells does not result in the formation of good spheroids.

Morphology of T47D spheroids

Microscopic image of multiple T47D spheroids growing in culture and at different seeding densities

500–10,000 T47D cells were seeded for spheroid generation and brightfield images of spheroids at Day 2, Day 4 and Day 7 were captured using EVOS XL microscope under 4x magnification. Scale bar denotes 200 μm.