Nexcelom Bioscience’s Cellometer K2 Image Cytometer enables handling of complex primary samples with an easy-to-use, simple process, which includes the following steps:

  • Pipette 20 µL
  • Insert slide
  • Select assay and click count
  • View results in 60 seconds
Cellometer K2

 

Simple, Automated Cell Counting in 60 Seconds

Using dual-fluorescence imaging and bright-field imaging, the Cellometer K2 detects and counts individual cells in a quick and accurate way. The Cellometer K2 automatically calculates and reports cell count, diameter, concentration, and percentage viability.

Users can load the sample, view the image, count the number of cells, and receive the results in less than 60 seconds.

With the Cellometer K2, users can:

  • Enhance accuracy
  • Increase throughput
  • Optimize consistency
  • Make sure all data is properly captured
  • Avoid miscounts, judgment errors, user-to-user variability, and interference from red blood cells (RBCs)
  • Count complex cells (irregular-shaped and clumpy cells)

Primary Cell Analysis: PBMCs, Hepatocytes and More

The Cellometer K2’s design has been specifically improved for analyzing primary cells from bone marrow, cord blood, peripheral blood, and other difficult samples used in an extensive range of research areas, such as:

  • Stem cells for cellular therapy
  • Nucleated cells for transplantation
  • Tumor cell suspensions for oncology
  • Splenocytes for vaccine development
  • PBMCs for immunology

Dual-color fluorescence enables live and dead nucleated cells to be stained, thus producing accurate viability results even when RBCs, platelets and debris are present. Since the K2 cell counter can accurately analyze both “clean” and “messy” samples, it can assess samples at a range of points throughout the processing of the sample, from initial collection, through separation, to cryopreservation.

The Cellometer K2 cell counter includes assays for analyzing an extensive array of primary samples, including the following:

Cellometer K2

Hepatocytes.

Cellometer K2

Jurkat.

Cellometer K2

Splenocytes.

Cellometer K2

PBMC.

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?

Dual-color fluorescence is strongly suggested to ensure accurate viability analysis for primary cells. This is because bright-field cell counting cannot differentiate nucleated cells from non-nucleated ones, and trypan blue staining cannot be detected as easily as fluorescent staining. Provided with standard assays, the Cellometer K2 can perform dual-fluorescence analysis of primary cells stained with acridine orange/propidium iodide (AO/PI).

The AO/PI Method

AO, a nuclear staining (or nucleic acid binding) dye, is permeable to live as well as dead cells. It produces green fluorescence by staining all nucleated cells. On the other hand, PI can enter only dead cells that have compromised membranes. It produces red fluorescence by staining all dead nucleated cells.

Since cells stained with both AO and PI tend to fluoresce red because of quenching, all dead nucleated cells fluoresce red and all live nucleated cells fluoresce green.

Cell-Based Assays: Cell Cycle, Apoptosis, and GFP

Cell Cycle

Cellometer K2

 

Nexcelom Bioscience’s Cellometer K2 Image Cytometer can perform primary cell-based assays like apoptosis, cell cycle, and green fluorescent protein (GFP) population analysis. Image cytometric analysis data can be exported from De Novo Software to FCS Express for data analysis and presentation to perform these cell-based assays.

To analyze cell cycle, Nexcelom Bioscience’s cell cycle kit can be used to analyze different cell cycle phases, to identify the S, SubG1, G2/M, and G0/G1 phase cell population. To analyze apoptosis, Nexcelom Bioscience’s Annexin V-FITC/PI and Caspase 3/8 staining kit can be used to find out the percent apoptotic cell population. Cellometer K2 can also be used to directly measure the GFP expression percent population.

 

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

Cellometer K2

 

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%

 

Cellometer K2

 

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 RBCs, Platelets or Debris

The dual-fluorescence AO/PI method involves using nuclear staining dyes that have an affinity to bind to nucleic acids contained in the cell nucleus. Since a majority of the mature mammalian RBCs lack nuclei, a fluorescent signal is generated only by live and dead mononuclear cells. The need for lysing RBCs is avoided, thereby saving time and removing an additional sample preparation step. Platelets, RBCs, and debris are not counted in the fluorescent channels.

Advantage of Fluorescent Counting for Primary Cells

The images given below illustrate the benefit of performing fluorescent counting for primary cells. The bright-field image depicts the combination of RBCs, nucleated cells, and platelets contained in the sample. Only the live and dead nucleated cells have been imaged 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

 

Different amounts of RBCs were used for spiking fresh human peripheral blood mononuclear cells (PBMCs). Counting of all cells (RBC + nucleated) was performed in the brightfield channel. Subsequently, counting of the nucleated cells was performed in the green fluorescent channel. The nucleated cell count was not affected by the different amounts of RBCs (1.8%, 4.6%, and 8.9%).

The bright-field image below (left side) shows a number of RBCs. In the fluorescent image (right side) illustrating cells stained with nuclear staining dye, the RBCs are not visible.

Cellometer K2

 

Cell Images for Data Verification

No two cells are the same

The Cellometer K2 Image Cytometer can be used to immediately visualize the cell morphology on-screen in the bright-field image.

To verify whether cells in the sample are being visualized and analyzed accurately, counted cells are specified on-screen. It is possible to view the bright-field counted images for trypan blue viability and basic cell counting.

It is also possible to view fluorescent counted images that indicate the number of counted live and dead nucleated cells, for dual-fluorescence primary cell viability assays.

The Cellometer K2 enables users to confirm whether:

  • Platelets, RBCs, and debris are being eliminated from results
  • Cells are accurately counted, based on shape and size
  • Cells inside clumps are counted individually
Cellometer K2

 

Cellometer K2

 

From the bright-field image, it can be seen that smaller debris is not counted but individual cells within pairs are counted. Live counted cells have been circled in green in the combined fluorescent counted image, while dead counted cells have been circled red.

  • It is possible to archive and export cell images for further use in presentations and publications.
  • Re-counting of saved images can be performed using either user-optimized or default analysis settings

Cellometer Primary Hepatocyte Viability Analysis Method

Conventional manual counting techniques can be inaccurate and time-consuming because hepatocytes are fragile in nature, have variable morphology, and tend to clump. Due to the tendency of hepatocytes to lose viability over time, variable or extended counting times can lead to inconsistent and inaccurate viability determinations.

Moreover, because of flow-induced shear stress, hepatocytes are too fragile to assess through flow cytometry. The most reliable technique to determine the viability of hepatocytes is cellometer image cytometry.

Dual-Fluorescence Staining Procedure

To determine viability, 20 µL of hepatocyte sample is blended with 20 µL of Cellometer AO/PI Staining Solution. Since DNA in all nucleated cells is stained by the AO dye, green fluorescence can be easily produced and the hepatocytes can be easily differentiated from debris.

DNA in all cells with compromised cell membranes is stained by PI to produce red fluorescence. The green fluorescence in cells stained with both PI and AO is absorbed by the red fluorescence through fluorescence resonance energy transfer (FRET), which makes all dead hepatocytes to fluoresce red, thus enabling them to be easily counted. The process is rapid, accurate, and gentle.

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).

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

As soon as mixing is performed, 20 µL of the stained sample is loaded onto the Cellometer Counting Chamber and inserted into the Cellometer K2 instrument. Imaging of the sample can be performed directly from the counting chamber.

The counting chamber is disposable, thereby avoiding the need to wash between samples and eliminating the risk of cross-contamination. Preconfigured parameters for primary hepatocytes are used for visualizing and analyzing the samples.

Cellometer K2

 

Analysis of Clumpy and Irregular-shaped Cells

Including NCI-60 and clumpy MCF-7 Cells

NCI-60 includes a cluster of 59 human cancer cell lines (originally 60) created by the National Cancer Institute to carry out screening.

  • All the 59 NCI-60 cell lines have been validated successfully on the Cellometer Image Cytometer.
  • Of these cell lines, 57% are clumpy, include debris, or exhibit huge variations in cell size or shape.

Cellometer Cell Counters are being used by all 40 of the NCI Comprehensive Cancer Centers.

Clumpy Cells

The MCF-7 breast cancer cell line can be extremely clumpy. Individual cells within these cell clumps are identified and counted by the Cellometer pattern-recognition software for accurate analysis (see figure below).

Cellometer K2

 

Irregular-Shaped Cells

Cells with irregular shape, like activated T-cells and RD cells, can be identified and counted by adjusting the Cellometer cell roundness setting.

Cellometer K2

 

Cell Size Analysis and Size-Based Counting

The Cellometer K2 Image Cytometer has the capability to automatically generate a cell size histogram based on cell diameter.

Since individual cell size measurements are generated by the Cellometer, it is possible to overlay several samples on a single histogram, thus facilitating the change in cell diameter over time to be analyzed.

Cellometer K2

 

10x Faster than Manual Counting

A manual hemacytometer takes about 5 minutes to count 1 x 106 cells. At times, counting of live and dead cells takes twice the time. The Cellometer K2 Image Cytometer has the ability to quantify cell count and concentration of live and dead cells, as well as percentage viability, within just 60 seconds.

Cellometer K2

 

Cellometer Precision

By ensuring a %CV (coefficient of variation) of less than 10% for the measurement of fluorescent concentration and viability, the Cellometer K2 Image Cytometer delivers superior reproducibility. The data given in the table below is based on four preparations of Jurkat cells that were stained with PI — 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%

Improve Data Accuracy and Consistency

  • Judgment errors are avoided
  • Recording and calculation errors are eliminated
  • Counting time is reduced, thus enabling users to run more experiments
  • Interference from RBCs is eliminated
  • Wash steps are avoided

Imaging/Counting Chambers: No Washing or Contamination

The two independent enclosed chambers of the Cellometer Disposable Imaging Chambers have an accurately regulated height. A typical single-channel pipette is used to load 20 μL cell suspension onto the chamber. The cells are visualized by inserting the chamber into the Cellometer cell counter. Since the sample loading and analysis method is simple, it is well-suited for fragile cells.

Cellometer K2

 

Following are the various significant benefits offered by the disposable Cellometer Cell Counting Chambers:

  • Controlled sample volume
  • Washing is not needed — time is saved
  • Chambers with larger depth to accommodate large cells
  • Cross-contamination is prevented
  • Most cost-effective, automated counting consumables
  • Biohazard risk to users is minimized

Dedicated On-line and On-site Applications Support

Expert technical support specialists from Nexcelom are available from 8:30 am to 5:00 pm EST to offer support via phone and online. They can assist with the following:

  • Installing the K2 Cell Viability Counter
  • Troubleshooting
  • Creating new cell types
  • Optimizing counting parameters
  • Training novice users

The Cellometer K2 software screen is provided with a help button at the bottom right. This button offers users immediate access to:

  • On-line tutorials and training videos
  • Submission of a Support Ticket
  • Software features and instructions

All Applications Specialists from Nexcelom are totally focused on using Cellometer Image Cytometry to perform image-based cell concentration and viability and cell-based assays.

Field-based Applications Specialists from Nexcelom also assist with:

  • Training
  • On-site demonstrations
  • Technical seminars
  • Troubleshooting