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Counting with the Countess

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to count cells using the Countess Cell Counter.

Reagents and Materials

  • Trypan Blue
  • Cell Solution

Things to Note before starting

Issues with concentration

The results from the countess are most accurate when the reading is between 1e6 and 30e6 cells / mL. For readings above and below that range, try to either dilute or concentrate your sample to get a reading within this range.

Estimated Timing

This takes ~2 minutes per sample you want to count.

Procedure

  1. Turn on the Countess
  2. Resuspend the cells in an appropriate volume based on the size of the cell pellet you have. 1
  3. Aliquot 10uL of the cell solution into a 96 well.
  4. Aliquot 10uL of Trypan Blue into the same well. Mix by pipetting up and down.
  5. Take up 10uL of the Cell & Trypan Blue solution and place into a Countess Slide. 2
  6. Insert the slide into the countess.
  7. On the screen, you will see the countess focus and adjust the brightness level. Wait until it finishes focusing (ie you cannot see anymore changes on the screen).
  8. Press the capture button.
  9. It should display the Total, Live, and Dead cells. Record these values.

Next Steps

Troubleshooting

  • If it shows that all or most of your cells are dead.
    • Do you see a lot of dots on the screen?
      • If so your sample might be too concentrated and the machine is looking at the higher concentration and calling them dead cells. Double check the slide under the microscope to ensure that all the cells have white interiors. If so - reinsert the cell and press capture again. Sometimes magically, the numbers will show all ALIVE.

Appendix

Footnotes

  1. This will be something that gets better with experience. I basically have 4 main resuspension volumes. If the pellet is not visible, resuspend in 200uL. If the pellet looks small, resuspend in 1mL. If the pellet is medium sized, resuspend in 10mL. If the pellet looks large, resuspend in 50mL. ↩

  2. Steps 2-4 should use a p20 and all use the same tip. ↩