See Application Notes and Article

CHM-900-0004: NucleoCounter Concept (mammalian cells)
CHM-900-0300: NucleoCounter YC-100 (yeast cells)

The NucleoCounter^® Concept

ChemoMetec A/S has developed and patented a new technology which addresses the problems of conventional cell counting. Compared to traditional cell counting methods, the operation of the NucleoCounter^® is fast, efficient and reliable. The NucleoCounter^® offers an analysis time of less than 30 seconds per sample, as well as high precision and reproducibility, by removing the opportunity for human error. The result is a fast and operator-independent cell count. The NucleoCounter^® is portable and compact, requires no daily cleaning or calibration, and is maintenance-free. This compact instrument fits perfectly in any mammalian cell laboratory (e.g. research, quality control,
or monitoring of production).

Features:

• Compact and user friendly design
• Bottle overfilling prevented by level detection sensor
• Overflow protection of pump and electronics by means of a hydrophobic filter at the pump inlet
• Control knob for infinitely vacuum setting (range: -300 mbar to -600 mbar)
• High resistant quick couplings (PVDF) for easy tubing connections
• Automatic vacuum control
• Optional pump exhaust tubing connector (prevents aerosol contamination of working environment)
• VACUBOY hand operator with 40 mm stainless steel adapter included
• All parts in contact with fluids, such as bottle, lid, tubings, connectors and hand operator,
  are autoclavable

Operation is as easy as 1 - 2 - 3.

The NucleoCounter^® is very easy to operate with simple procedures for:

1. Performing the sample preparation
2. Simple sampling with the NucleoCassette
3. One touch analysis performed on the Run keypad

1-Sample Preparation

Mix a representative cell sample with equal volumes of
reagent A-100 (lysis/disaggregation buffer) and reagent B (stabilizing buffer).

2-Sampling

Load the NucleoCassette with the lysate solution by immersing the tip of the cassette into the solution and pressing the piston.

3-Analysis

Place the NucleoCassette in the instrument and press the "Run" key. After 30 seconds the cell count is presented on the instrument display. Or the data can be transferred to an external PC using a USB connection, or printed on an
external printer.

Key benefits of the NucleoCounter^®

Easy operation for multiple users - as simple as 1-2-3

Very little time is required for training of operators and no continuous attention is required

30 sec. analysis time

Calibration free:

• No calibration to cell type - the variation in cell size and DNA content of mammalian cells is within the range of the NucleoCounter.
• No calibration to volume - the volume of sample measured in every single analysis is known, and therefore no calibration to volume is necessary. The thickness of the measurement chamber of each NucleoCassette is measured during production and coded with black dots on each Cassette. The NucleoCounter reads the code. During production the NucleoCounter is calibrated for its viewing area, which will remain unchanged throughout the lifetime of the instrument. The product of the two factors (area multiplied by thickness) gives an accurate sample volume.
• Durable optical components make the NucleoCounter calibration free through out its lifetime.

No cleaning
The NucleoCassette contains the entire flow system (pump, tubing, mixer, reagents and waste container) as well as the measurement chamber. As the NucleoCassette is discarded after use, no cleaning or maintenance of the NucleoCounter is needed.

Safe sample handling and disposal
After analysis, the sample and the PI are contained inside the cassette, which can be safely discarded.

Maintenance and service free

• No maintenance is required, as the NucleoCounter is made of very durable and stable components. The optics, mechanics and electronics have negligible thermal and aging drift properties. The light source and the linear actuator have been tested to last for >1 x 10E6 samples.
• •No service is needed as no calibration, cleaning nor replacement of components is necessary.

Portable/compact
The weight of the NucleoCounter is 3 kg and the size is: 38 x 26 x 22 cm (W x H x D) which makes it easy to move between laboratories or sites.

Excellent reproducibility
Results from different NucleoCounters can be directly compared:

• All NucleoCounters are calibrated to respond as the same Master NucleoCounter at ChemoMetec.
• The initial calibration will be maintained throughout the lifetime of the NucleoCounter.

Objective cell count
Automation eliminates the subjective judgment of the operator. The NucleoCounter cell count is therefore an objective and operator-independent count.

Small sample volume
Only about 50 μl of sample mixture is loaded into the NucleoCassette. It is possible to perform an accurate and precise estimate of the cell concentration using only a very small amount of sample.

Large analysis volume
2 μl, which is 10-20 times the volume in a haemocytometer, is analyzed in one measurement.

Declumping
Cell aggregates are dissolved by lysing cells with Reagent A-100 and B before the total cell count, improving the quality of results.

Documentation
NucleoView software is ideal for additional documentation purposes.

NucleoView Software
The NucleoView software can be used for additional documentation of analysis data, for example:

• MF (Multiplication Factor caused by the actual dilution of the sample by: Pre dilution and Reagent A100 and B)
• Viable or Non-Viable analysis
• Operator ID
• Sample comment
• In addition, the Raw Image is presented on the display.

Components of the System:

The NucleoCounter

The NucleoCounter is the first commercially available instrument based on ChemoMetec's technique. The principle of the technique is the well-known method of fluorescence microscopy, implemented in a small, highly advanced fluorescence microscope, combined with a CCD camera and image analysis software. The fluorescence microscope is designed to detect signals from the fluorescence dye propidium iodide, which stains the DNA of cell nuclei. This result represents either a total or non-viable cell concentration, depending on the preparation of the cell sample. Detected signals are correlated to a cell count, which is presented to the user on the built in display. The image and results can also be transferred to a PC for viewing and documentation purposes, using the NucleoView™ software application.

The enclosure of the instrument contains an aluminium profile which holds all the essential parts for the fluorescence microscope and the computing components, which perform the image analysis for counting of the cells in the sample. Apart from the fluorescence microscope, the instrument also incorporates a printer port, USB port, DC input voltage port, a Keyboard and a Display.

NucleoCassette
Safe sample handling and disposal

The NucleoCassette is a highly specialised device, which holds the key to several of the unique features of the NucleoCounter. The fluorescent dye propidium iodide (PI) is immobilized inside the flow channels of the disposable cassette. When the cassette has been loaded with approximately 50 µl of sample mixture, the PI is dissolved and mixed with the sample, staining the nuclei of the mammalian cells.

After placement in the NucleoCounter the stained mixture is automatically transferred to the measurement chamber where the fluorescent image is recorded. The measurement volume can be as large as 2 µl.

After analysis, the sample and the PI are contained inside the cassette, which can be safely discarded. This offers a unique method for safe sample handling and disposal.

As the NucleoCassette contains the entire flow system as well as the measurement chamber, neither cleaning nor maintenance of the NucleoCounter instrument itself is needed.

NucleoCounter reagents

To determine the total amount of mammalian cells in a sample, the use of reagent A-100 and Reagent B is necessary.

The lysis Reagent A-100, with a pH of approximately 1.25, is used to disrupt the plasma membranes of the mammalian cells, rendering the nuclei susceptible to staining with propidium iodide (PI). In addition to this, the reagent contributes effectively to disaggregation of cell clusters.

The stabilizing Reagent B is used after Reagent A-100 to raise the pH value, allowing PI to stain the DNA of the cells more efficiently.

It is recommended to use equal volumes of sample, Reagent A-100 and Reagent B (e.g. 200 ml of each)

Propidium Iodide (PI)

The fluorescent dye propidium iodide (PI) stains the DNA of cell nuclei. The PI intercalates with the DNA, with 1 dye molecule per 4-5 DNA base pairs.

As little as about 2.8 μg of PI is immobilized in each NucleoCassette. As PI stains the cell nuclei it needs to be permeable to the dye. The dye cannot penetrate a viable cell, thus it is necessary to lyse the cell membrane prior to staining, when counting the total amount of cells.

Non-viable cells, on the other hand, are permeable and can therefore be stained directly with the PI and counted by the NucleoCounter. Combining these two results makes it possible to determine the cell viability of a sample

Cell viability – how it works:
The cell viability of a sample is determined using the total cell count and the count of non-viable cells (see the figure below). When a sample is mixed with Reagent A-100 and Reagent B, a lysis of the viable cell membrane takes place rendering all the cell nuclei susceptible to staining with propidium iodide (PI) resulting in a total cell count. Non-viable cells are permeable without treatment and are therefore stained directly with PI, resulting in a non-viable cell count.

Cell Species – more information:
Mammalian cells can differ with respect to many physical and/or morphological properties. Many methods for the counting of mammalian cells are therefore dependent on a calibration or adjustment in order to reliably count the number of cells present in a sample. Since the principle used in the NucleoCounter for the counting of mammalian cells is based on staining the DNA within the nuclei, it becomes evident that the method is reliable in the measurement of various cell species without any adjustment or calibration. The method has been successfully applied for the counting of heterogeneous cell lines such as adipocytes, CHO cells and hybridomas.

Application Notes and Article

Application Note - Cell Counts and Viability.pdf

Application Note - Cell Counts and Viability Reagent A100.pdf

Application Note - Dilution of the Samples.pdf

Application Note -Microcarrier.pdf

Article from BIOforum Europe Magazine.pdf