Counting Cells with Tryple Reagent and Countess II FL Automated Cell Counter

The Countess II FL Automated Cell Counter is a benchtop instrument designed and manufactured by Invitrogen, which performs cell counting and viability measurements in concert with trypan blue stain.  The Countess offers fast, easy and accurate cell counting without using a hemocytometer, thus eliminating the tedium and subjectivity of manual cell counting.

GIBCO brand TrypLE Select and TrypLE Express are recombinant serine protease enzymes of high purity, which contain no components of animal origin.  TrypLE is designed to remove adherent cells from the surface of plastic flasks and plates in a gentler manner.  In addition, it is room temperature stable and a direct replacement for trypsin.

• This protocol details the procedure for obtaining accurate cell counts using TrypLE cell dissociation products with the Countess II FL Automated Cell Counter.

1. Warm TrypLE to 37°C.
2. Aspirate growth medium from the flask (or plate), rinse the cells with 5 mL of Dulbecco’s Phosphate Buffered Saline (D-PBS) without calcium and without magnesium, and gently rock the vessel back and forth.  Decant D-PBS.
3. Add an appropriate volume of pre-warmed TrypLE Select or TrypLE Express to the flask (i.e., 1 mL in a T25 cm2 flask, 3 mL in a T75 cm2 flask).  Gently rock the vessel, allowing the solution to coat cell the sheet completely.
4. Incubate at 37°C until the cells have visibly detached (observe at 5 min intervals).  Gently tap the vessel to dislodge any cells that remain adhered to the cell surface.
5. Add an appropriate volume of complete growth media, re-suspend cells and pipette repeatedly to break up any clumps that may be present.

1. Push Power to start the instrument.
2. Change the mode to Cell mode prior to counting cells by pressing Settings and then Cells
3. Press Parameters to ensure appropriate settings.
4. Take 10 μL of cell suspension from step 5, add to 10 μL of 0.4% trypan blue stain.  Mix gently by pipetting the liquid up and down.
5. Add 10 μL of the sample mixture into the half-moon shaped chamber port on one side of Countess cell counting chamber slide.
6. Insert slide, sample side A first into the slide port of the Countess until a soft click is heard.  Samples can be counted from one chamber (A) or both chambers (A and B).
7. Press Zoom button and use the focus knob to optimize image for analysis:
   
   • Live cells have bright centers and dark edges.
   • Dead cells have uniform blue color throughout the cell with no bright centers.
8. Use the grid to navigate the slide and check the image in several fields.  If desired, lock focusing using Focus lock knob.
9. Press Count cell button to obtain results.
10. Record cell count or save after reading.
11. Press more data button to see data details as well as graphical representation of data.
12. Remove and discard the slide appropriately.

For detailed instructions using Countess automated cell counter, refer to the manual on thermofisher.com/countess