Cellometer Auto 2000 Cell Viability Counter

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Introduction:

Simple, Automated Cell Counting in 30 Seconds

Simple, User-friendly Procedure

Pipette 20µl

Insert Slide

Select Assay & Click Count

View Results in 30 seconds!

The Auto 2000 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 < 30 seconds

The Auto 2000 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

User-Friendly Touch Screen

The user-friendly touch screen displays pre-optimized assays for common cell types. User-specific protocols can be easily created and saved to the menu. The simple, pre-optimized assays make the instrument easy to operate and simplify training for new users.

With the click of a button, researchers can select a new assay with saved counting parameters, making the instrument ideal for labs testing a variety of cell types. Auto-save options allow users to quickly test a series of samples.

Primary Cell Analysis: PBMCs, Stem Cells, and more

The Cellometer Auto 2000 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 Auto 2000 to evaluate samples at a variety of points throughout sample processing - from initial collection, to separation, to cryopreservation.

The Cellometer Auto 2000 features one-touch assays for analysis of a wide range of primary samples, including:

Nucleated Cells from Whole Blood

PBMCs / Splenocytes following Separation

Stem Cells

Tumor and Tissue Cell Suspension

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 Auto 2000 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.

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.

Cell Images for Data Verification

No two cells are the same.

With the Auto 2000 Cell Counter, 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

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 Cell Counter

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 Auto 2000 Cell Counter 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.

For experiments involving SF9 or SF21 cells, saved images can be viewed and re-counted to analyze changes in morphology over time.

The minimum and maximum cell diameter settings can be optimized to count specific cells in a sample.

This example demonstrates counting of mature dendritic cells cultured from PBMCs based on cell diameter.

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 Auto 2000 Cell Viability Counter calculates cell count and concentration for live and dead cells and % viability in just 30 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 Auto 2000 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
A	4	4.20E+06	91.1	10%	2%
B	4	1.06E+06	22.7	7%	1%
C	4	3.27E+06	57.5	7%	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

Auto-Save Data and Cell Images

The Auto 2000 allows users to generate specific folders to which data reports and images can be auto-saved. Using the auto-save feature ensures all data is accurately captured in the correct location. It also enables users to test a series of samples very quickly.

Users can configure the Auto 2000 to request a new sample name for each sample that is imaged and counted or apply auto-numbering to individual samples in a series to further reduce analysis time. Users can also apply a time stamp to individual data files.