Cellometer K2 Fluorescent Viability Cell Counter
Fluorescent automated cell counter for complex primary samples
Why Cellometer K2?
- Primary Cell Analysis
- Live/Dead Nucleated Cell Counts
- Cell Based Assays: Cell Cycle, Apoptosis, GFP
The Cellometer K2 is fast and very accurate. We love that the dye is nuclear to ensure accuracy of the cell counts. Sure beats counting by hand!
The Cellometer K2 is great and easy to use. It gives clear images quickly. We have been using it for a couple years and never have any problems with it!
The Cellometer K2 is very fast and efficient. It has turned what used to be a hassle in the lab into a breeze!
Our customers include:
Introduction to the Cellometer K2 Fluorescent Viability Cell Counter
Simple, User-friendly Procedure
Pipette 20µl |
Insert Slide |
Select Assay & Click Count |
View Results in 60 seconds! |
Simple, Automated Cell Counting in 60 Seconds
The Cellometer K2 utilizes bright field imaging and dual-fluorescence imaging to quickly and accurately identify and count individual cells. Cell count, concentration, diameter, and % viability are automatically calculated and reported.
Load Sample, View Image, Count Cells, and Obtain Results in < 60 seconds
The K2 Allows Users to:
- Increase throughput
- Increase accuracy
- Improve consistency
- Ensure all data is correctly captured
- Count difficult cells (clumpy, irregular-shaped)
- Eliminate judgment errors, miscounts, interference from red blood cells and user-to-user variability
Primary Cell Analysis: PBMCs, Hepatocytes, and more
The Cellometer K2 is specifically optimized for analysis of primary cells from peripheral blood, cord blood, bone marrow, and other complex samples for use in a wide range of research areas, including:
- Nucleated Cells for Transplantation
- PBMCs for Immunology
- Splenocytes for Vaccine Development
- Stem Cells for Cellular Therapy
- Tumor Cell Suspensions for Oncology
Dual-color fluorescence allows for staining of live and dead nucleated cells, generating accurate viability results even in the presence of debris, platelets, and red blood cells. Accurate analysis of both ‘messy’ and ‘clean’ samples enables the K2 to evaluate samples at a variety of points throughout sample processing – from initial collection to separation, to cryopreservation.
The Cellometer K2 features assays for analysis of a wide range of primary samples, including:
Hepatocytes |
Jurkat |
Splenocytes |
PBMC |
Live / Dead Nucleated Cell Counts using Dual-Fluorescence
Green fluorescent live cell image
Red fluorescent dead cell image
Why Dual-Fluorescence?
Because bright field cell counting does not differentiate nucleated from non-nucleated cells and trypan blue staining is not as easy to detect as fluorescent staining, dual-color fluorescence is strongly recommended for accurate viability analysis for primary cells. The K2 is equipped with standard assays for dual-fluorescence analysis of primary cells stained with AO/PI.
The AO/PI Method
Acridine Orange, AO, is a nuclear staining (nucleic acid binding) dye permeable to both live and dead cells. It stains all nucleated cells to generate green fluorescence. Propidium iodide, PI, can only enter dead cells with compromised membranes. It stains all dead nucleated cells to generate red fluorescence. Cells stained with both AO and PI fluoresce red due to quenching, so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red.
Cell-Based Assays: Cell Cycle, Apoptosis and GFP
Cell Cycle
Cellometer K2 Image Cytometer has the ability to perform basic cell-based assays such as cell cycle, apoptosis, and green fluorescent protein (GFP) population analysis. These cell-based assays can be performed by exporting image cytometric analysis data to FCS Express from De Novo Software for data analysis and presentation.
For cell cycle analysis, the different cell cycle phases can be analyzed using the cell cycle kit from Nexcelom Bioscience to determine the SubG1, G0/G1, S, and G2/M phase cell population. For apoptosis analysis, using Annexin V-FITC/PI and Caspase 3/8 staining Kit from Nexcelom Bioscience to determine percent apoptotic cell population. GFP expression percent population can also be directly measured using Cellometer K2.
| Cellometer K2 | SubG1 | G0/G1 | S | SG2/M |
|---|---|---|---|---|
| AVE | 1.0% | 51.1% | 13.6% | 31.1% |
| STD | 0.4% | 1.6% | 1.0% | 1.1% |
Apoptosis
Untreated (Negative Control)
| Healthy | Apoptotic | Necrotic | Debris | |
|---|---|---|---|---|
| AVE | 81.9% | 8.1% | 4.0% | 6.0% |
| STD | 1.6% | 1.3% | 0.4% | 1.1% |
| CV | 1.9% | 16.1% | 9.3% | 18.3% |
Treated (Positive Control)
| Healthy | Apoptotic | Necrotic | Debris | |
|---|---|---|---|---|
| AVE | 58.8% | 24.7% | 13.8% | 2.7% |
| STD | 1.9% | 1.1% | 1.2% | 0.2% |
| CV | 3.2% | 4.3% | 9.0% | 9.2% |
No Interference from Red Blood Cells, Platelets, or Debris
The dual-fluorescence AO/PI method utilizes nuclear staining dyes that bind to nucleic acids in the cell nucleus. Because most mature mammalian red blood cells do not contain nuclei, only live and dead mononuclear cells produce a fluorescent signal. There is no need to lyse red blood cells, saving time and eliminating an extra sample preparation step. Red blood cells, platelets, and debris are not counted in the fluorescent channels.
The advantage of fluorescent counting for primary cells
These images (right) demonstrate the advantage of fluorescent counting for primary cells. The bright field image shows the combination of nucleated cells, red blood cells, and platelets present in the sample. Only the live and dead nucleated cells are visualized and counted in the green and red fluorescent channels.
| Sample | Measurement | Total nucleated | All | RBC | % RBC | n |
|---|---|---|---|---|---|---|
| PBMC+RBC | Mean
CV |
1.26E+07
6.2% |
1.39E+07
8.8% |
1.23E+06 | 8.9% | 10 |
| PBMC+1/2RBC | Mean
CV |
1.21E+07
4.8% |
1.27E+07
5.4% |
5.82E+05 | 4.6% | 10 |
| PBMC+1/4RBC | Mean
CV |
1.22E+07
7.9% |
1.24E+07
7.3% |
2.27E+05 | 1.8% | 10 |
Fresh human PBMCs (peripheral blood mononuclear cells) were spiked with varying amounts of RBCs (red blood cells.) All cells (nucleated + RBC) were counted in the brightfield channel. Nucleated cells were then counted in the green fluorescent channel. Varying amounts of RBCs (1.8%, 4.6%, and 8.9%) did not affect the nucleated cell count.
Several red blood cells are indicated in the bright field image (above, left). The red blood cells are not visible in the fluorescent image (above, right) detecting cells stained with nuclear staining dye.
Cell Images for Data Verification
No two cells are the same.
With the Cellometer K2 Image Cytometer, cell morphology can be immediately viewed on-screen in the bright field image.
Counted cells are indicated on-screen for further verification that cells in the sample are being imaged and analyzed properly. Bright field counted images can be viewed for basic cell counting and trypan blue viability.
Fluorescent counted images indicating counted live and dead nucleated cells can be viewed for dual-fluorescence primary cell viability assays.
Users can confirm that:
- cells are counted correctly, based on size and shape
- cells within clumps are being counted individually
- red blood cells, platelets, and debris are being excluded from results
The bright field image confirms that individual cells within pairs are being counted and smaller debris is not being counted. In the combined fluorescent counted image, live counted cells are circled in green. Dead counted cells are circled in red.
- Cell images can be archived and exported for use in publications and presentations.
- Saved images can be re-counted using default or user-optimized analysis settings
Cellometer Primary Hepatocyte Viability Analysis Method
Due to hepatocytes’ variable morphology, fragile nature, and tendency to clump, traditional manual counting methods can be time-consuming and inaccurate. Because hepatocytes lose viability over time, extended or variable counting times can generate inaccurate and inconsistent viability determinations. Hepatocytes are also too fragile to evaluate using flow cytometry due to flow-induced shear stress. Cellometer image cytometry is the most reliable method for determination of hepatocyte viability.
Dual-fluorescence Staining Procedure
For viability determination, 20µl of hepatocyte sample is mixed with 20µl of Cellometer AO/PI Staining Solution. The acridine orange (AO) dye stains DNA in all nucleated cells, generating green fluorescence and easily differentiating hepatocytes from debris. Propidium iodide (PI) stains DNA in all cells with compromised cell membranes, generating red fluorescence. In cells stained with both AO and PI, the green fluorescence is absorbed by the red fluorescence via FRET (fluorescence resonance energy transfer), so all dead hepatocytes fluoresce red and can be easily counted. The procedure is fast, gentle, and accurate.
Bright field image (left) shows the variable morphology of primary hepatocytes. Dual fluorescence image (right) shows counted live hepatocytes (circled in green) and counted dead hepatocytes (circled in red).
Cellometer Analysis
Immediately after mixing, 20µl of stained sample is loaded into the Cellometer Counting Chamber and inserted into the Cellometer K2 instrument. The sample is imaged directly from the counting chamber. Because the counting chamber is disposable, no washing is required between samples and there is no risk of cross-contamination. Samples are imaged and analyzed using pre-set parameters for primary hepatocytes.
Analysis of Clumpy and Irregular-shaped Cells
Including NCI-60 and clumpy MCF-7 Cells
NCI-60 is a group of 59 human cancer cell lines (originally 60) developed by the National Cancer Institute for screening purposes.
- 57% of the NCI-60 cell lines are clumpy, contain debris, or display large variations in cell shape or size
- All 59 NCI-60 cell lines have been successfully validated on the Cellometer Image Cytometer
All 40 of the NCI Comprehensive Cancer Centers use Cellometer Cell Counters.
Clumpy Cells
The MCF-7 breast cancer cell line can be very clumpy. The Cellometer pattern-recognition software identifies and counts individual cells within these cell clumps for accurate analysis (shown above).
Irregular-shaped Cells
The Cellometer cell roundness setting can be adjusted for recognition and counting of irregular-shaped cells, such as RD cells and activated T-cells.
Cell Size Analysis & Size-based Counting
The Cellometer K2 Image Cytometer Automatically generates a cell size histogram based on cell diameter.
Because Cellometer generates individual cell size measurements, multiple samples can be overlaid on one histogram enabling analysis of the change in cell diameter over time.
10x Faster than Manual Counting
Counting 1 x 106 cells takes approximately 5 minutes with a manual hemacytometer. Counting live and dead cells sometimes takes twice as long. The Cellometer K2 Image Cytometer calculates cell count and concentration for live and dead cells and % viability in just 60 seconds.
Improve Data Accuracy & Consistency
- Eliminate Wash Steps
- Eliminate Judgment Errors
- Eliminate interference from RBCs
- Eliminate Recording & Calculation Errors
- Reduce Counting Time … Run More Experiments
Cellometer Precision
The Cellometer K2 Image Cytometer offers excellent reproducibility, with a %CV (Coefficient of Variation) of <10% for fluorescent concentration and viability measurements. The data (right) is based on four preparations of Jurkat cells stained with propidium iodide, a fluorescent nuclear-staining dye.
| Sample | N Value | Average Live Cell Concentration | % Viability | CV of Concentration | CV of Viability |
|---|---|---|---|---|---|
| Jurkat | 24 | 3.61E+06 | 92.2% | 8.9% | 1.0% |
| Human PBMC | 10 | 5.94E+06 | 96.0% | 4.7% | 0.5% |
| Mouse Splenocyte | 10 | 1.86E+07 | 88.6% | 5.6% | 0.7% |
Imaging / Counting Chambers: No Washing or Contamination
Cellometer Disposable Imaging Chambers consist of two independent enclosed chambers with a precisely controlled height. Cell suspension of 20 microliters is loaded into the chamber using a standard single channel pipette. The chamber is inserted into the Cellometer cell counter and the cells are imaged. This simple sample loading and analysis method is ideal for fragile cells.
The disposable Cellometer Cell Counting Chambers offer several key advantages:
- Time savings – no washing
- No risk of cross-contamination
- Reduced biohazard risk to users
- Controlled sample volume
- Large-depth chambers for large cells
- Most affordable automated counting consumables
Dedicated On-line and On-site Applications Support
Experienced Nexcelom Technical Support Specialists are available from 8:30 am to 5:00 pm EST for phone and online support and can assist with:
- Creation of new cell types
- Optimization of counting parameters
- Troubleshooting
- Training of new users
- Installation of the K2 Cell Viability Counter
The help button at the bottom right of the Cellometer K2 software screen gives users instant access to:
- Software features and instructions
- On-line tutorials and training videos
- Submission of a Support Ticket
All Nexcelom Applications Specialists are 100% focused on image-based cell concentration & viability and cell-based assays using Cellometer Image Cytometry.
Nexcelom Field-based Applications Specialists are also available for:
- On-site demonstrations
- Training
- Troubleshooting
- Technical Seminars
Applications for Cellometer K2 Fluorescent Viability Cell Counter
Adipocytes
Automatically measure cell size of freshly isolated adipocytes and plot size histogram. DNA staining fluorescence dyes are used to identify cells from lipid droplets. »
Adoptive Cell Transfer Therapy
Use Cellometer to perform cell based assays and measure cell size, viability and concentration of cell lines and primary samples used in adoptive cell therapy research. »
Apoptosis
Automatically detect and analyze Caspase3 and 8, JC-1, and Annexin V apoptotic events using the Cellometer image cytometery. »
Image Cytometry for Cell Cycle Analysis
Automatically measure the cell cycle of mammalian cells. Generated cell cycle histogram allows for easy data analysis and presentation. »
Cell Viability Measurement Using Trypan Blue or AO/PI
When should you use trypan blue and when should you use acridine orange/propidium iodide to measure cell viability? »
Cell Viability and Necrosis
Cell viability is performed using various fluorescent membrane exclusion dyes, such as PI, EB, 7AAD, and others. This assay is performed by enumerating cells in captured bright-field and fluorescent images. And Necrotic cells are detected using propidium iodide. »
TNC Concentration & Viability for Clinical (Blood) Samples
Analyze fresh and processed blood and bone marrow samples without lysing: no interference from RBCs. »
Cell Size Assay
Performing cell size measurement assay and using cell size to count cells within preset cell size parameters. For adipocytes, stem cells, Sf9 cells, dendritic cells, and others. »
NCI-60 Cancer Cell Lines
Automatically measure live cell concentration and viability of cancer cell lines used in oncology research and most of all biology research. »
Quantitative Measurement of GFP Transfection
Rapidly identify fluorescence positive cells from a sample, analyze individual cell fluorescence intensity, calculate cell concentration, size and determine the GFP transfection automatically. »
Fresh & Cryo Preserved Primary Hepatocytes
Automatically measure live hepatocyte concentration and viability using dual fluorescent nuclear stains, for human, rat, mouse and horse. »
Immunology Research
Automatically quantify cell viability and concentration for a variety of immunologically relevant samples such as: bone marrow, cord blood, slpenocytes, lymphocytes, isolated mononuclear cells, tumor digests, murine samples, and others. »
Insect Cells
Automatically measure live cell concentration, viability for baculovirus infected insect cells. Cell size histogram live cell concentration and viability are generated within less than 60 seconds using 20 µl sample. »
Peripheral Blood Mononuclear Cells (PBMC)
Automatically measure live cell concentration and viability without lysing red blood cells for consistent results from patient samples. Other cells include splenocytes and bone marrow. »
WBCs in Whole Blood
Automatically measure nucleated cell concentration without lysing red blood cells using nuclear staining dyes (AO), for human and mouse blood. »
Performance of the Cellometer K2 Image Cytometer
Total Cell Concentration Range of Jurkat Cells Measured by Cellometer K2
Concentration Dynamic Range Figure 1 depicts the dynamic range for cell concentration measurements on Cellometer K2. This data set was taken on a concentration series of cultured Jurkat cell line.
Samples from 1 x 105 – 1 x 107 cells/ml can be counted without further dilution.
The %CV at each concentration was below 10%.
Figure 1: Table of results for cell concentration dynamic range
Cellometer K2 Repeatability and Consistency
| Sample | N Value | Average Live Cell Concentration | % Viability | CV of Concentration | CV of Viability |
|---|---|---|---|---|---|
| Jurkat | 24 | 3.61E+06 | 92.2% | 8.9% | 1.0% |
| Human PBMC | 10 | 5.94E+06 | 96.0% | 4.7% | 0.5% |
| Mouse Splenocyte | 10 | 1.86E+07 | 88.6% | 5.6% | 0.7% |
Figure 2: Table of results for cell concentration and viability using AO/PI
The results indicate the accuracy of the Cellometer K2 instrument in assessing the viability of Jurkat cells using AO/PI for cell viability. Jurkat, human PBMC, mouse splenocytes were tested at 24, 10, and 10 sample replications, respectively. The viability average was calculated and plotted. The results show the reliability and accuracy of the Cellometer K2 in measuring cell concentration and viability of mammalian cells.
Consistency and Accuracy Comparison to Hemacytometer
Jurkat
| N = 20 | Hemacytometer | Cellometer K2 |
|---|---|---|
| Average | 1.03E+06 | 1.04E+06 |
| STDEV | 6.60E+04 | 5.57E+04 |
| %CV | 6.4% | 5.3% |
5 µm beads
| N = 20 | Hemacytometer | Cellometer K2 |
|---|---|---|
| Average | 1.07E+06 | 1.03E+06 |
| STDEV | 5.90E+04 | 5.38E+04 |
| %CV | 5.5% | 5.2% |
The Cellometer K2 is fast and very accurate. We love that the dye is nuclear to ensure the accuracy of the cell counts. Sure beats counting by hand!
The Cellometer K2 is great and easy to use. It gives clear images quickly. We have been using it for a couple years and never have any problems with it!
The Cellometer K2 is easy to use and great for counting cells that are prone to clumping together. It can’t cell culture without this machine!