The assessment of how many cells have been successfully transfected or transduced in a cell population is a basic and critical evaluation parameter in many cell and molecular biology labs. Commonly, the cells of interest are transfected or transduced with a construct that results in the expression of a fluorescent protein (FP) reporter, such as GFP. The Invitrogen Countess II FL Automated Cell Counter loaded with your choice of Invitrogen EVOS light cubes allows a quick and simple method to easily obtain transfection or transduction efficiency data. The procedures described below can be applied to any commonly used FP reporter system that is currently evaluated using microscopy or flow cytometry.


Materials

  • Countess II FL Automated Cell Counter (Cat. No. AMQAF1000)
  • Invitrogen Countess Cell Counting Chamber Slides (Cat. No. C10228) or Countess II FL Reusable Slide (Cat. No. A25750)
  • Appropriate EVOS light cube(s)
  • Fluorescent protein (FP)-expressing cells

Protocol—instrument setup

  1. Turn on the Countess II FL Automated Cell Counter and install appropriate EVOS light cube(s).
  2. Install the appropriate slide holder for either the disposable or reusable slide.
  3. Locate the slide(s) to be used.

Protocol—culture setup

  1. Acquire a eukaryotic cell suspension for FP expression analysis.
  2. If cell viability information is desired in addition to FP expression analysis, combine 10 μL of the sample with 10 μL of trypan blue. (Note: Trypan blue may quench the fluorescent signal, depending on the FP’s localization.)
  3. Apply 10 μL of the stained sample to the counting slide.
  4. Insert the counting slide into the Countess II counter’s sample port to initiate autofocus, then press “Capture”. Record the viability
  5. Once brightfield counting is complete, reinsert the sample slide and select the appropriate light source from the upper-right corner of the screen.
  6. Adjust the fluorescence excitation light intensities to minimize background.
  7. Press “Capture” once more.

Using the Countess II FL Automated Cell Counter and appropriate light cubes, what used to require multiple manual cell counts per sample with a hemocytometer can now be obtained in as little as 10 seconds with a single count on the Countess II FL Automated Cell Counter, while reducing the count-to-count variability between lab users. This results in better consistency between experiments and dramatic time savings. Figures 1 and 2 present data from common cell counting applications involving the analysis of transduction efficiency.

Figure 1. Determination of transduction efficiency using the Countess II FL Automated Cell Counter. A Countess II FL Automated Cell Counter was equipped with EVOS light cubes for DAPI (Cat. No. AMEP4650) and GFP (Cat. No. AMEP4651). HeLa cells were transduced with Invitrogen CellLight Mitochondria-GFP, BacMam 2.0 (Cat. No. C10600), and then stained with Invitrogen NucBlue Live ReadyProbes Reagent (Cat. No. R37605) for total cell staining. In this case, 72% transduction efficiency was determined for the cell sample in less than 10 seconds.

Figure 2. Fluorescent cell counts similar to those obtained by flow cytometry can be easily acquired with the Countess II FL Automated Cell Counter, with a single light cube. U2OS cells were transduced using Invitrogen CellLight Nucleus-GFP, BacMam 2.0 (Cat. No. C10602), and allowed to incubate for 36 hours. The cells were evaluated for fluorescent protein expression by (A) flow cytometry and (B) the Countess II FL Automated Cell Counter. The transduction percentages determined by the two methods are very close to each other.


Summary

Flow cytometry and manual cell counting with a hemocytometer and microscope are the gold standards for fluorescent protein expression analysis, but both require advanced user training and relatively expensive instrumentation. The Countess II FL Automated Cell Counter is able to provide comparable data, but with faster turnaround and greater cost-effectiveness while reducing user-to-user variability.