Nexcelom Bioscience
Cellometer K2 Image Cytometer Optimized Analysis of Primary Cells

The K2 is a PC-based cell imager for automated 4x bright field and dual-channel fluorescent image capture and analysis. The K2 accurately performs cell count, size, viability, intensity, and population analysis and achieves count and viability measurements 10x faster than manual methods. This instrument is ideal for cells lines and primary cells in complex samples characterized by heavy debris, red blood cell contamination, low cell concentrations, and multiple cell types, including PBMCs, stem cells, cancer cells, splenocytes, hepatocytes, and whole blood. In addition, the K2 can easily perform cell-based assays such as Apoptosis, Cell Cycle, and GFP analysis.

Simple, Automated Cell Counting in 60 Seconds

Simple, User-friendly Procedure

Simple, User-friendly Procedure

Pipette 20µl

Insert Slide

Select Assay & Click Count

View Results 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.

Results in less than 30 seconds 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

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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:

hepatocyte cells Jurkat cells

Hepatocytes

Jurkat

Splenocytes PBMC

Splenocytes

PBMC

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Live / Dead Nucleated Cell Counts using Dual-Fluorescence

Green fluorescent live cell image

Green fluorescent live cell image

Red fluorescent dead 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.

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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

1.26E+07

1.39E+07

1.23E+06

8.9%

10

 

CV

6.2%

8.8%

 

 

 

PBMC+1/2RBC

Mean

1.21E+07

1.27E+07

5.82E+05

4.6%

10

 

CV

4.8%

5.4%

 

 

 

PBMC+1/4RBC

Mean

1.22E+07

1.24E+07

2.27E+05

1.8%

10

 

CV

7.9%

7.3%

 

 

 

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.

Cell images showing the exclusion of red blood cells

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.

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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
Bright field counted cell image Fluorescent counted cell image

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

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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.


Hepatocyte Viability

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.

Cell Based Assays: Cell Cycle, Apoptosis and GFP

Cell Cycle

Cell Cycle data plot


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.

K2 SubG1 G0/G1 S G2/M
AVE 1.0% 51.1% 13.6% 31.1%
STD 0.4% 1.6% 1.0% 1.1%

Apoptosis

Apoptosis untreated (negative control)

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%


Apoptosis treated (positive control)

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%

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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

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).
Count-irregular-shaped cells

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.

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Cell Size Analysis & Size-based Counting

Cell Size Histogram

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.

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10x Faster than Manual Counting

Hemacytometer Under Microscope

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%

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Imaging / Counting Chambers: No Washing or Contamination

Cellometer disposable imaging chambers
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

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Dedicated On-line and On-site Applications Support

Experienced Nexcelom Technical Support Specialists are available from 8:30am to 5:00pm EST for phone and on-line 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
K2 help
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

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