Flexible dual fluorescence and brightfield cell counting with reusable slide compatibility

The Invitrogen Countess 3 FL Automated Cell Counter has fluorescence capabilities with two easily interchangeable fluorescent light cubes selected from 14 spectrum-spanning options. Like the Countess 3 instrument, the Countess 3 FL instrument can count cells and measure viability in brightfield, while also reporting fluorescent protein expression, apoptosis, transfection efficiency, and cell viability using fluorescent reagents in as few as 30 seconds.

 

Fluorescence cell counting also allows the use of fluorescence-based cellular assays that offer more specificity with complex samples by using fluorescent cellular stains or fluorescently tagged cells.


Features

The Countess 3 FL instrument shares common features with the Countess 3 model: It is accurate, precise, fast, simple, and automated; it offers viability measurement and single-cell data across a wide variety of cell lines; it is compatible with reusable slides; and it has optional software to support  21 CFR Part 11 compliance for electronic records. In addition, its fluorescence capabilities open up additional options and applications.

Fluorescence applications

Advanced viability, apoptosis, transfection efficiency, fluorescent protein expression, and more.

Interchangeable LED light cubes

Extend your choice of fluorescent proteins and dyes beyond GFP and RFP

Optimized Ready Count reagents

Designed for the Countess 3 FL cell counter


Fluorescence counting applications

In addition to counting cells, the fluorescence capabilities of the Countess 3 FL instrument are often used to assess cell viability, apoptosis, transfection efficiency, and fluorescent protein expression. But the applications extend to any cellular parameters that are autofluorescent or can be labeled using fluorescent dyes. You’ll find data illustrating cell culture quality control, CAR-T cell screening, CRISPR-Cas9 editing efficiency, and other fluorescence applications in Sample Data.

 

Cell viability. Assessing cell viability is a key step in daily cell culture and is required for accurate and efficient downstream processing. The Countess 3 FL fluorescence options offer a sensitive and specific viability check that can be performed quickly and easily prior to downstream sample analysis.

 

Although trypan blue is widely used for viability assessment in brightfield cell counting, it can alter cell morphology in certain cases. For example, when dead mouse PBMCs are stained with trypan blue, they can expand and sink to a different focal plane than live cells, complicating accurate detection and counting. As a fluorescence alternative, Invitrogen ReadyCount Green/Red Viability Stain contains acridine orange and propidium iodide (AO/PI) in an optimized combination. Fluorescent staining provides great sensitivity and greater specificity of live/dead status in a mixed cell population without altering the morphology of the cell.

Viability measurement using fluorescence

Murine PBMCs were stained with ReadyCount Green/Red Viability Stain and counted with the Countess 3 FL Automated Cell Counter. In fluorescence mode, this reagent’s pre-optimized AO/PI mixture results in greater live/dead specificity. Dead cells (38%) are stained red by the PI and are detected with the Texas Red 2.0 light cube, while all nucleated cells (99%) fluoresce green with the AO dye and are detected with the GFP 2.0 light cube.

Results screen with cell concentration of 1.02 million cells per milliliter and 38% dead cells as stained by propidium iodide.

Apoptosis detection

U2OS (human osteosarcoma epithelial) cells were incubated with 4 µM staurosporine to induce apoptosis, labeled with CellEvent Caspase 3/7 Green Detection Reagent and SYTOX Deep Red Nucleic Acid Stain, and counted on a Countess 3 FL Cell Counter equipped with GFP 2.0 and Cy5 2.0 light cubes. Analysis identified apoptotic (green, 83%), dead (red, 32%), and “dying” (teal, 16%) cells. Percentages sum to more than 100% because the 16% of cells that express both stains are counted in all three categories.

Results screen with cell concentration of 2.12 million cells per milliliter and 83% of the population in apoptosis.

Apoptosis detection. Fluorescent stains can target different aspects and stages of apoptosis, such as plasma membrane alterations (Annexin V), mitochondrial changes (JC-1), or caspase activation (Caspase 3/7). Pairing one of these with a viability stain on the Countess 3 FL can identify apoptotic as well as live and dead cells.

Transfection efficiency. Assessing the percentage of cells successfully transfected or transduced in a population is critical for research. Commonly, transfection or transduction is paired with a fluorescent protein reporter such as GFP. The Countess 3 FL Automated Cell Counter, loaded with your choice of light cubes, offers a quick and simple method to easily obtain transfection efficiency data.

Adherent GFP transfected cells
GFP transfected Jurkat cells counted in suspension with transfection efficiency measured by percent GFP positive

Transfection efficiency

Jurkat cells were transfected with a GFP expression vector. (left) Cells can be seen adhered to the vessel in this image captured on an EVOS M5000 Imaging System. (right) Cells were trypsinized and counted on a Countess 3 FL Cell Counter equipped with an EVOS GFP light cube. Analysis showed that 50% of cells were transfected successfully.

“We compared to a hemocytometer and the Countess II always obtained very close results. The fluorescent measurements are a great tool for us to quantify the % transfection and transduction efficiency in our experiments. The light cubes used to detect fluorescence are very easy to switch out when more than two fluorescence sources are needing to be detected. The automatic focusing is very effective and accurate with the option to manually focus.”

David Waynar,

Santa Cruz Biotechnology


EVOS LED light cubes

The Countess 3 FL instrument has two optional fluorescence channels, which will detect fluorescent colors determined by the insertion of individual light cubes. With 14 interchangeable light cubes to choose from, the Countess 3 FL instrument is not limited to detection of cells expressing GFP and RFP.

Array-of-EVOS-LED-light-cubes

Suppose you are interested in measuring both apoptosis and viability of cells in a population. You can use an apoptosis indicator that measures caspase activation, such as CellEvent Caspase-3/7 Green Detection Reagent along with the EVOS GFP 2.0 light cube. Additionally, use a viability dye such as SYTOX Red Dead Cell Stain with the EVOS Cy5 2.0 light cube to measure dead cells that have progressed past apoptosis. This staining combination, along with the two EVOS light cubes, offers a fast and simple multiplex method to obtain apoptosis and viability data.


Optimized ReadyCount reagents

Fluorescence staining can provide rich data on cells, even in heterogeneous samples, but can be difficult to optimize. Invitrogen ReadyCount reagents are bench stable fluorescent stains that are pre-optimized for use with Countess 3 Automated Cell Counters.

ReadyCount Red Dead Cell Stain

This reagent stains dead cells and can be detected with an EVOS RFP 2.0 or Texas Red 2.0 light cube.

ReadyCount Blue Nuclear Stain

This reagent stains nucleic acids and can be detected with an EVOS DAPI 2.0 light cube.


Videos and demos


Specifications

 

Technical specifications Processing time: <30 seconds
Sample concentration range: 1 x 10⁴–1 x 10⁷ cells/mL
Particle diameter range: ~4–60 µm
Cell diameter range: ~4–60 µm
Required sample volume: 10 μL
USB drive: 32 GB (FAT32-, exFAT-, or NTSF-compatible)
Optics: 5 megapixel camera, 2.5X optical magnification
Physical characteristics Instrument dimensions:
9" (W) x 5½" (D) x 9" (H); 228.6 mm (W) X 139.7 mm (D) x 228.6 mm (H)
Weight: 8 lbs; 17.6 kg
Counting area

Area: 2.26 mm x 1.69 mm (3.82 mm²)

Graphic image depicting the area of a typical standard hemocytometer and the area counted by the Countess 3 Cell counter which is significantly larger

Included with instrument USB drive for storing and transferring data to your computer
Countess Disposable Slide Holder
Countess Reusable Slide Holder
Countess Cell Counting Chamber Slides (Qty 50)
Power cord and 4 plug adaptors to accommodate most countries
Quick reference card (QRC)
Sold separately Countess Reusable Slide
High-Power USB Wi-Fi Module (dongle)
Countess Cell Counting Chamber Slides
Trypan Blue Stain
Countess SAE Software Solution
Countess 3 Cell Counting Standard Slide

Ordering information

* Extended waranty included without additional cost

A custom quote will be provided after selecting from the following items:
  • Countess 3 FL Automated Cell Counter
  • Countess 3 FL Reusable Slide
  • Countess Cell Counting Chamber Slides (Qty 50)
  • Countess Cell Counting Chamber Slides (Qty 500)
  • Invitrogen Trypan Blue Stain (0.4%)
  • EVOS Light Cube, GFP 2.0
  • EVOS Light Cube, RFP 2.0
  • EVOS Light Cube, DAPI 2.0
  • ReadyCount Green/Red Viability Stain
  • 3 years Rapid Exchange Extended Warranty

For research use only. Not for use in diagnostic procedures.