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Enumerating Spheres in Suspension Cultures

Counting spheres, such as mammospheres, tumorspheres and neurospheres, doesn't have to be challenging. This video shows a simple technique for enumerating cells cultured as spheres:

For a detailed protocol on counting spheres, please see below:

  1. Harvest the spheres from each well of a 6-well plate and put them into a 14 mL tube.
  2. Rinse each well with 2 mL DMEM-F12 and combine with the collected suspension.
  3. Centrifuge at low speed, approximately 100 – 200 x g for 5 minutes.
  4. Take the suspension and carefully aspirate the supernatant so as not to disturb the sphere pellet.
  5. Gently resuspend the pellet in 500 uL of the complete medium per well of a 6-well plate.
  6. Using a fine-tipped marker, divide one well of a 96-well plate into quadrants by drawing a plus sign on the underside of the well.
  7. Add 50 uL of the sphere suspension to the well and view the spheres under the microscope
  8. Count the spheres using a hand-tally counter. To ensure the accuracy, count at least 50 spheres in the well. If you count fewer than 50 spheres, gently mix the sphere suspension to ensure even distribution and count again using a larger volume. Calculate the sphere concentration by dividing the count by the counting volume. Calculate the total sphere count by multiplying the concentration by the total volume.

Due to potential sphere and cell aggregation, this method provides only an estimate of the total number of spheres formed in culture.

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