Maintenance of MDA-MB-231 cells before spheroid generation

After thawing from liquid nitrogen, cells were maintained in Nunclon Delta T25 cell culture flasks in Gibco DMEM 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 290 g for 3 minutes and placed in the incubator. This is Day 0.
  7. On Day 1, 100 μl of complete medium containing 6 μg/ml collagen I was added to each well, so that the final concentration of collagen in the medium is 3 μg/ml. The plate was centrifuged at 100 g for 3 minutes and placed back in the incubator.
  8. Spheroids were ready on Day 4 (>2,000 cells) to Day 7 (<2,000 cells) depending on the seeded cell number.

Skip to Cell viability assay protocol or 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.
  • On Day 1, add the medium carefully through the sides of the wells.
  • On Day 1, the medium should be at room temperature and not at 37 degrees. Also, keep the collagen I on ice until ready to use.
  • We have observed that centrifuging the plate after seeding cells facilitates cell aggregation leading to uniform spheroid formation consistently. However, this step is optional.

Morphology of MDA-MB-231 spheroids

Microscopic image of multiple MDA-MB-231 spheroids growing in culture following different seeding densities

1,000–10,000 MDA-MB-231 cells were seeded for spheroid generation and brightfield images of spheroids at Day 2, Day 4 and Day 7 were captured using EVOS M5000 microscope under 4x magnification. Scale bar denotes 400 μm.

ECM makes a difference in MDA-MB-231 spheroid formation

Microscopic image of multiple MDA-MB-231 spheroids growing in the presence of collagen and at different seeding densities

1,000–10,000 MDA-MB-231 cells were seeded for spheroid generation with or without collagen according to the above-mentioned protocol. Brightfield images of cell aggregates and spheroids on Day 4 were captured using EVOS M5000 microscope under 4x magnification. Scale bar denotes 400 μm. The addition of collagen was found to dramatically improve the formation of spheroids.

Characterization of MDA-MB-231 spheroids

Assessing cell viability using PrestoBlue-HS cell viability reagent

  1. On Day 4 and Day 7, cell viability assay was performed using the PrestoBlue HS cell viability reagent.
  2. The PrestoBlue HS reagent was warmed up to room temperature. Then, 20 μl was added to each well containing 200 μl of medium using a multichannel pipette, and gently mixed by pipetting 2–3 times. Wells containing only fresh medium and the reagent were used as normalization control (blank).
  3. The plate was incubated at 37°C for 6 hours.
  4. Following this, the plate was centrifuged at 290g for 3 minutes, and fluorescence was read using the Varioskan LUX Multimode Microplate Reader using the following settings:
  • Excitation: 560 nm; Emission: 590 nm
  • 12 nm excitation bandwidth
  • Measurement time: 100 ms
  • Bottom optics reading
  • “Circle” selection in the multipoint reading
  • Instrument temperature: 37°C

The data was exported to Microsoft Excel for analysis. Individual fluorescence values were normalized to Blank and mean of at least 6 replicates per cell seeding number were plotted using graphing and statistics software. The experiment was repeated 3 times.

line graph showing PrestoBlue HS fluorescence as a function of the number of cells seeded per well
Graph showing relative viabiltiy of MDA-MB-231 spheroids of Day 4 and Day 7 post–cell seeding. Error bar represents SEM.

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

  1. The population of live and dead cells on Day 4 and Day 7 MDA-MB-231 spheroids were visualized by staining them using the LIVE/DEAD Viability/Cytotoxicity kit.
  2. Working solution was prepared by adding Calcein AM and EthD-1 at a final concentration of  of 1 μM and 2 μM respectively in fresh medium (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. Spent medium of spheroids was changed 1:1 with working solution and incubated at 37°C for 3 hours.
  4. Following this, they were washed twice with PBS + 5% FBS (1:1 change), and finally resuspended in PBS + 0.5% FBS (1:1 change), each time centrifuging the plate at 290g for 3 minutes at room temperature to settle the spheroids and minimize background during fluorescence imaging.
  5. The spheroids were then imaged using CellInsight CX7 high-content screening platform under 4X objective. Each image is a maximum intensity projection of 15 μm z-stacks (12-15 for 1,000–2,000 cells, 15-25 for 5,000–10,000 cells). 

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 spheroids

Representative images of Day 4 (left panel) and Day 7 (right panel) of MDA-MB-231 spheroids showing the live (green staining) and dead (red staining) cell population. Blue indicates nuclear staining. Scale bar = 300 µm.