Maintenance of cells before PC-3 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 2 passages before seeding for spheroid generation. ATCC protocol was followed for subculturing.

Materials required

Protocol for spheroid generation

  1. Once the flask was 70-80% confluent, 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 and cell suspension was diluted in ice cold DMEM medium.
  5. Geltrex matrix was then thawed completely on ice and added at a final concentration of 3% to the above cell suspension. (for lower cell densities reduce the concentration of Geltrex matrix)
  6. A volume of 200 μl of the cell suspension was added to each well of 96-well Sphera plates.
  7. Plates were then centrifuged at 1,500 rpm for 10 min at 4°C and placed in the incubator at 37°C and 5% CO2.
  8. Day 5-9, spheroids were ready, 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

  • Be very careful about the temperature conditions of Geltrex matrix. Keep it ice-cold at all times.
  • While preparing the cell suspension make sure the media is cold. This cold media doesn’t harm the cells.
  • Make sure after adding Geltrex matrix into the suspension it is mixed properly without forming bubbles.
  • 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 PC-3 spheroids cultured in the presence of Geltrex matrix

Microscopic image of multiple PC-3 spheroids growing in the presence of Geltrex matrix and at different seeding densities

150–5,000 PC-3 cells were seeded using Geltrex matrix for spheroid generation and brightfield images of spheroids at Day 3, Day 5, Day 7 and day 9 were captured using EVOS M7000 microscope under 4x magnification. Scale bar denotes 650 μm.

Morphology of PC-3 spheroids cultured in the absence of Geltrex matrix

Microscopic image of multiple PC-3 spheroids growing in the absence of Geltrex matrix and at different seeding densities

150–5,000 PC-3 cells were seeded without using Geltrex matrix for spheroid generation and brightfield images of spheroids at Day 3, Day 5, Day 7 and day 9 were captured using EVOS M7000 microscope under 4x magnification. Scale bar denotes 650 μm.

Characterization of PC3 spheroids

Assessing cell viability using PrestoBlue-HS cell viability reagent

  1. Spheroids were generated following the spheroid generation protocol above. On Day 5, 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 4 hours.
  4. Following this, fluorescence was read using the Varioskan LUX multimode plate reader using the following settings: 
  • Excitation: 560 nm; Emission: 590 nm 
  • 12 nm bandwidth 
  • Measurement time: 100 ms 
  • Bottom reading of plate 
  • Instrument temperature: 37°C 

The data were 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.

bar graph showing PrestoBlue HS fluorescence as a function of the number of PC-3 cells seeded per well

Graph showing relative viabiltiy of PC-3 spheroids of Day 5 post–cell seeding. Error bars represent SEM.

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

  1. The populations of live and dead cells in Day 5 PC-3 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  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 1,500 rpm for 5 minutes at room temperature to settle the spheroids) to minimize background during fluorescence imaging.
  5. Fluorescence images of the spheroids were captured using the CellInsight CX7 high-content screening platform under 4X objective.
microscopic views of fluorescently stained PC-3 spheroids
Representative images of Day 5 PC-3 spheroids showing the live (green fluorescence) and dead (red fluorescence) cell populations (top panel). The bottom panel shows nuclear staining. Scale bar = 100 μm.
bar chart showing the percent dead cells for each seeded cell sample
Graph showing percent dead cells in PC-3 spheroids on Day 5. Fluorescence reading was captured using Varioskan LUX multimode plate reader. Values were plotted using GraphPad Prism. Spheroids treated with 70% methanol overnight were used as positive control for dead cells for respective seeding densities.