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

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 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.

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

Tips

  • 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.
  • 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 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.

Characterization of T47D spheroids

Assessing cell viability using PrestoBlue HS cell viability reagent

  1. Spheroids were generation following the spheroids generation protocol above. On Day 6, 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 incubation, the plate was centrifuged at 250g for 5 minutes, and fluorescence was read using the Varioskan LUX multimode plate 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

Relative viability of T47D spheroids of Day 6 post–cell seeding. Error bars represent SEM.

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

  1. The population of live and dead cells in Day 6 T47D 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 4 μM each 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 2 hours.
  4. Following this, they were washed once with PBS + 5% FBS (1:1 change), and finally resuspended in PBS + 0.5% FBS (1:1 change, each time centrifuging the plate at 250g for 5 minutes at room temperature to settle the spheroids) to minimize background during fluorescence imaging.
  5. Fluorescence reading of the spheroids were acquired using Varioskan LUX multimode plate reader (Ex/Em 530+12/645+12 nm for EthD-1) and the spheroids were then imaged using CellInsight CX7 high-content screening platform under 4X objective. Each image is a maximum intensity projection of 12 μM z-stacks (18-20 for 500–1,000 cells, 25-30 for 2,000–4,000 cells, 30–35 for 6,000-8,000 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 6 T47D spheroids showing the live (green staining) and dead (red staining) cell population (top panel). The bottom panel shows nuclear staining. Scale bar = 200 μM. The nucleus has been pseudocolored for better visibility.

bar chart showing the percent dead cells for each seeded cell sample

Percent dead cells in T47D spheroids on Day 6. Fluorescence reading was captured using Varioskan LUX multimode plate reader and analyzed in Microsoft Excel. Spheroids treated with 70% methanol overnight were used as positive control for dead cells for respective seeding densities.