Performance and application data for Countess Automated Cell Counters

Performance data shows that Countess 3 Cell Counters produce accurate and precise counts across a range of cell types and challenges. Application data shows the kind of output researchers can expect in studies of viability, apoptosis, transfection efficiency, CAR-T cell culture, CRISPR transduction, and more.

 

For additional application data, download the application notes on this page as well as in Resources.

Cell counting accuracy and precision

Machine-learning algorithms result in highly accurate and precise cell counts, comparable with flow cytometer counts.

CHO-K1, HeLa, HEK293, Jurkat, and human PBMCs (across a range of sizes) were counted using the Attune NxT Flow Cytometer (purple bars), a Countess 3 FL Automated Cell Counter (red bars), and a hemocytometer and microscope (manual counting, gray bars). The Countess 3 FL instrument and hemocytometer bars represent an average of 6 independent counts and are highly consistent. Error bars (standard deviation for the counts) are wider for manual counts than for Countess 3 counts or flow cytometry.

Machine-learning algorithms result in highly accurate and precise cell counts with less variability than manual counts.

CHO-K1, HeLa, HEK293, Jurkat, and human PBMCs (across a range of sizes) were counted using a Countess 3 FL Automated Cell Counter (blue dots) and manually using a hemocytometer and microscope (green dots). Medians (short horizontal lines) were similar between Countess 3 and hemocytometer counts, but individual hemocytometer counts (dots) showed far more variability—more than 20% across different users.

Challenging cell types

Machine-learning algorithms result in highly accurate cell counts with challenging cell types and with better precision than manual counting.

 

Human and murine peripheral blood mononuclear cells (PBMCs), notoriously challenging to count due to their small size and low contrast, were counted (top) using the Countess 3 FL Automated Cell Counter (blue bars) and manually using a hemocytometer and microscope (green bars). All bars represent six counts. The error bars represent standard deviations, which are significantly larger for manual counts. Images (bottom) show human (left) and murine (right) PBMCs identified and counted.

Clumped cells

Machine-learning algorithms generate accurate counts with clumpy cells and sample debris. 

 

The Countess 3 FL Automated Cell Counter was used in brightfield mode to count RAW (mouse macrophage) cells, which are small and prone to clumping. Trypan blue staining was used to establish viability. The figure shows both the raw image (left) and the enhanced image (right), in which live cells are contoured green and dead cells red. The counting algorithm was able to resolve the cells in the clumps and properly segment and count the cells. Debris was recognized and automatically excluded from counts.

Viability

Viability of murine PBMCs in brightfield and fluorescence mode. 

 

The Countess 3 and Countess 3 FL Automated Cell Counters were used in brightfield and fluorescence mode to count murine PBMCs, which can be as small as 5 µm. In the brightfield count (left), trypan blue stain (0.4%) was used to distinguish viability. The Countess 3 cell counter encloses live (translucent) cells with green contours (circles) and dead (deeply stained) cells with red contours to indicate their viability. 

 

In fluorescence mode (right), the acridine orange and propidium iodide (AO/PI) assay results in greater specificity of live/dead status. The AO stains all nucleated live cells green and can be detected with the EVOS GFP 2.0 light cube, while the PI stains the dead cells red and can be detected with the EVOS Texas Red 2.0 light cube. This is one case when fluorescence staining is a more accurate viability measure than trypan blue.

Apoptosis

Apoptotic and dead cell detection in fluorescence mode. 

 

After incubation with 2 and 4 µM staurosporine, USO2 (human osteosarcoma epithelial) cells were labeled with 1:400 CellEvent Caspase-3/7 Green Detection Reagent to identify apoptotic cells, and then stained with 1:1,000 SYTOX Red Dead Cell Stain to denote all dead cells. Cells were incubated at room temperature for 30 minutes and counted on a Countess 3 FL Automated Cell Counter equipped with EVOS LED Light Cubes, GFP 2.0 and Cy5 2.0. Both green and far-red signal increased with higher drug doses, indicating significant increases in apoptosis and cell death, respectively. Percentages for 4 µL staurosporine sum to more than 100% because the 16% of cells that express both stains are counted in all three categories.

Precise single cell counting and viability assessment with Countess

Explore our latest application note focusing on the counting of single cells and nuclei for scRNA-Seq and scATAC-Seq.  Enhance your research with precise cell counting and viability assessment. 

 

Single-cell RNA sequencing (scRNA-Seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-Seq) technologies enable new insights into gene expression and regulation by providing data resolution at the single-cell level. To obtain high-quality single-cell data, protocols for scRNA-Seq and scATAC-Seq require an accurate count of viable suspended single cells or nuclei as input, with minimal presence of cellular aggregates and dead cells. The Invitrogen Countess 3 FL Automated Cell Counter is a dependable solution for accurately counting cells and nuclei, evaluating their viability, and assessing aggregation. To help ensure optimal performance of the Countess 3 FL cell counter in such protocols, this application note provides a best practices guide.

Accurate and automated counting of single cells and nuclei for scRNA-Seq and scATAC-Seq. 

 

Counting of nuclei on the Countess 3 FL Automated Cell Counter using FL-based counting resulted in accurate counts, verified via sequencing. FL-based counts of nuclei isolated from the GM12878 cell line (A) and PBMCs (B) counted more nuclei than BF-based or Countess II instrument counts. (C) Isolation of nuclei from brain tissue produced accurate FL-based counts, comparable to counts from the Cellaca™ MX High-Throughput Automated Cell Counter. (D) Next-generation sequencing (NGS) confirmed accurate loading of 2,000 target nuclei, obtained from FL-based counts. *FL-based counts available on Countess 3 FL instrument using software update 3. Data obtained by 10x Genomics R&D.

Transfection efficiency

With the Countess 3 FL Automated Cell Counter, you can quickly verify what proportion of your cells have been transfected or transduced successfully before moving on to downstream experiments and analysis.

Quantification of transfection efficiency in fluorescence mode.

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

Transduction efficiency measured by flow cytometry vs automated cell counting. 

 

U2OS cells were transduced using Invitrogen CellLight Nucleus-GFP, BacMam 2.0 and allowed to incubate for 36 hours. The cells were evaluated for fluorescent protein expression by (graph, left) flow cytometry and (image, right) a Countess II FL Automated Cell Counter with a GFP light cube. The transduction percentages determined by the two methods are almost identical—49.5% vs 49%.

Cell culture quality control during CAR-T cell expansion

When expanding T cells for research and development of new immunotherapies such as CAR-T, it’s important to quickly assess T cell health in a small sample of cells to help ensure maximum viability. The Countess 3 FL Automated Cell Counter can help provide fast, accurate measurements of cell viability and concentration both before and after isolation and activation, helping save time and precious cells for future experiments.

CAR-T cell culture quality control. 

 

CAR-T cells were isolated using the Dynabeads Untouched Human T Cells Kit and activated using Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation. The cell culture was monitored before and after expansion using brightfield counting with trypan blue staining on the Countess 3 FL Automated Cell Counter. In images (left) and histograms (right), the T cells and beads are differentiated automatically, with T cells appearing as live (green) and beads as dead (red). Beads can also be gated out manually based on size and intensity, if desired.

Viability and transduction efficiency of CRISPR-Cas9 transduced cells

In genome editing experiments using CRISPR-Cas9, lentiviral transduction is often used to help deliver the CRISPR-Cas9 complex efficiently into the cells. Control particles that express a fluorescent protein (such as GFP), and potentially knock out other genes (such as HPRT), are frequently used to measure transduction efficiency. The Countess 3 FL Automated Cell Counter can be used to quantitate both viability and transduction efficiency in these cells.

Lentiviral transduction efficiency measured by GFP expression. 

 

U2OS cells expressing the Cas9 protein were transduced with LentiArray CRISPR Positive Control Lentivirus, human HPRT, with GFP and a negative control lentivirus (scramble, GFP) at a multiplicity of infection (MOI) of 1 and 2. Two days later, cells were counted on the Countess II FL cell counter for GFP expression. (left) Display of representative counting results on the Countess II FL instrument. (right) Bar graphs show the percentages of cells positive for GFP, indicating successful transduction.

Sample and staining verification for flow cytometry and cell sorting

Many flow cytometry and cell sorting protocols recommend a specific cell concentration or range for optimal staining and analysis. A preliminary pass with a Countess 3 Automated Cell Counter prior to flow cytometry or fluorescence-assisted cell sorting (FACS) can help increase experimental success and potentially save valuable time, money, and sample. You can quickly verify that (a) you have enough cells to complete the protocol, (b) you have used the optimal amount of staining reagent, (c) you haven’t lost significant numbers of cells during staining, and (d) your cells have been stained or transduced effectively.

Effect of cell concentration on cell cycle analysis by flow cytometry.

Different concentrations of live Jurkat cells were labeled with a constant concentration (10 μM) of Vybrant DyeCycle Orange Stain, a cell-permeant dye that fluoresces on binding to DNA and produces a “chair-shaped” cell cycle histogram when stained optimally. The same concentration of stain produced poor cell cycle histograms with both low (left) and high (right) cell concentrations, while staining with the optimal cell concentration of 1 x 10⁶ cells/mL (center) produced the proper cell cycle histogram. Staining histograms can be verified quickly and easily with a Countess 3 FL Automated Cell Counter equipped with an EVOS LED Light Cube, RFP 2.0.

 

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