How Tali® Works

The Tali® Image Cytometer is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that performs suspension cell-based assays, including:

• GFP and RFP expression
• Annexin V apoptosis
• Cell viability analysis

Using only 25 μL of sample volume, Tali® Image Cytometer takes 10–75 seconds for a typical cell population analysis.  In addition to a bright field channel, the Tali® Image Cytometer features two fluorescent channels (green and red), enabling it to simultaneously count green and/or red fluorescent stained cells, as well as cells expressing GFP and RFP (Figure 1). The Tali®Image Cytometer also presents comprehensive data with graphic reports and allows the export of data for archiving and sample comparisons.

The Tali® Image-Based cytometer is perfect for:

• Rapid analysis of GFP/RFP vector transfection efficiency
• Identification of GFP and/or RFP positive cells
• Simple two color apoptosis assays
• Accurate cell viability population analysis
• Quick verification of samples prior to sorting or high-end flow cytometry analysis

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Figure 1. Simultaneous analysis of GFP and RFP expressing cells. The Tali® Image-Based Cytometer displays two fluorescent channels (green and red), enabling it to simultaneously count green and red fluorescent proteins and stains. It also provides actual cell counts and automatically calculates cell concentration for each cell population.

With the Tali® Image Cytometer, running simple experiments, visualizing cells and obtaining cell measurements is fast, easy and convenient. Its small footprint can also easily fit into your lab’s workspace.

• Accurate—Obtain statistically significant three-parameter population analysis.
• Fast—Analysis and cell counting in typically less than 1 minute per sample.
• Versitile—Generate visual and analytical data for GFP/RPF expression, apoptosis, and cell viability.
• Convenient—Setup in minutes with no cleaning or routine maintenance.

The Tali® Image Cytometer incorporates image-based cell counting and fluorescence detection algorithms to perform the assays listed below for cells in suspension.

GFP/RFP Analysis
The Tali® Image Cytometer counts and calculates the relative amounts in the sample of cells expressing
GFP or RFP, as well as any unstained or non-fluorescent cells. The GFP/RFP assay menu also includes combined GFP/RFP and viability assays. Use the Tali® Viability Kit –Dead Cell Red with cells expressing green fluorescent proteins and use the Tali® Viability Kit – Dead Cell Green with cells expressing red fluorescent proteins. (Figure 2)

Viability
The Tali® Image Cytometer determines the number and proportion of viable and dead cells using the Tali® Viability Kit –Dead Cell Red, which stains the dead cells red or the Tali® Viability Kit – Dead Cell Green, which stains the dead cells green.

Apoptosis
The Tali® Image Cytometer distinguishes between apoptotic, dead, and live cell populations using the Tali® Apoptosis Kit, counts the cells in each population, and calculates relative amount of each population in the sample. (Figure 3)

Quick Count
The Tali® Image Cytometer also provides quick and accurate cell counts without the need for staining your cells and determines the concentration of your sample and the average cell size.

While we have developed the assays above specifically for use on the Tali® Image-Based cytomer, any fluorescent dye or protein that fits into the green (FITC, Alexa Fluor® 488, GFP, etc) or red (RFP, PI, etc..) fluorescent channels can also be analyzed by the Tali® Image Cytometer.

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Figure 2. Population analysis of GFP and RFP expressing cells. The Tali® Image Cytometer GFP+RFP display is shown after measuring U2OS cells transduced with CellLight® Nucleus-GFP and CellLight® Plasma Membrane-RFP BacMam. Representative images of cell fluorescence are shown on the left side of the screen while quantitative results showing population numbers (%GFP positive, %RFP positive, %GFP and RFP positive, and %GFP and RFP negative) are shown on the right.

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Figure 3. Apoptosis analysis using Annexin-Alexa Fluor® 488 and propidium iodide.  The Tali® Image Cytometer Apoptosis display after measuring Jurkat cells stained with the Tali® Apoptosis Assay kit. Representative images of cell fluorescence are shown on the left side of the screen while quantitative results showing population numbers (Live, Dead, and Apoptotic) are shown on the right.

The Tali® Image Cytometer scans the Tali® Cellular Analysis Slide, collecting bright-field and fluorescence images from up to 20 different fields of view (each containing approximately 5,000 cells for a 1 x 10⁶ cells/mL sample). Using a highly sensitive camera and a sophisticated image-processing and analysis algorithm, the Tali® cytometer is able to capture the faintest signals in a quantitative manner and displays the relevant data (e.g., cell count vs. cell size, cell count vs. fluorescence) in tables and histograms.

• See Tali® Image Cytometer Specifications

While sharing the same basic workflow and user interface, all Tali® assays differ in the fluorescence channels being collected and analyzed by the internal algorithm. The number of fields to be collected (4, 9, 13, 18, or 20) can be selected by the user prior to each run on the touch screen interface. In each field of view, the Tali® cytometer captures a combination of brightfield and fluorescence images, designed specifically for the assay selected. After capturing all fields of view, the instrument uses sophisticated digital image analysis algorithms to calculate the total cell population and determine the fluorescence characteristics of those cells on a cell-by-cell basis. The algorithm in the Tali® cytometer is expressly designed to distinguish single cells from clumps and to determine the number of cells within a clump based on the area of the clump (up to 60 μm in diameter) and the average cell size in the population being measured. The ability of the Tali® Image Cytometer to accurately determine the number of cells within a clump is unique to this image-based system.

Relevant data (e.g.,cell count vs. cell size, cell count vs. fluorescence) are displayed in both table and histogram formats, allowing users to: select specific fields of view for review; zoom in on selected fields of view; display channel-specific layers (i.e., images captured in bright-field, green fluorescence, and red fluorescence channels or a combination of the three channels); and gate on cell counts by cell size and/or fluorescence intensity (where applicable). Analysis data, including the image files, can be downloaded to a USB flash drive any time after an assay has been run, and transferred to a computer for sample comparisons or additional analysis.

• Find Detailed Information in Tali® Image Cytometer FAQs

The intuitive user interface of the Tali® Image Cytometer makes it extremely easy to collect data and analyze results in as few as six easy steps:

1. Select an assay.
2. Add cells to the slide.
3. Insert the slide into the Tali® cytometer.
4. Focus the image of the cells.
5. Press “Run Sample”.
6. Collect and analyze data.

Typical processing times for the Tali® Image Cytometer are between 10 and 120 seconds, depending on the number of fields that are being captured and the complexity of the assay chosen. Following analysis, both qualitative (.bmp) and quantitative (.csv) data can be transferred to your computer using a USB drive.

The accuracy of results generated by the Tali® Image Cytometer was compared with those of a high-end flow cytometer. For each assay (apoptosis, cell viability, RFP/GFP expression) and cell type (JKT, HEKn, 293MSR, U2OS, CHO-S) tested, results from the Tali® cytometer were comparable to those from the flow cytometer (Figures 5–8).

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Figure 5. Comparison of cell viability as analyzed on the Tali® Image Cytometer and a flow cytometer. For end-point assays, cell viability was measured for five different cell types (Jurkat, U2OS, 293MSR, HEKn, and CHO-S). Cells were stained using the Tali® Viability Kit and then measured using either the Tali® cytometer or a flow cytometer. For all cell types tested, viable cell population data from the Tali® Image Cytometer were comparable to those obtained by flow cytometry.

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Figure 6. Time-course comparison of cell viability as analyzed on the Tali™ Image Cytometer and a flow cytometer. Population data obtained with the Tali® cytometer are comparable to those obtained using a flow cytometer. For the time-course assay, 10% (v/v) ethanol was added to 293MSR cells to induce cell death. Starting at time 0 and every 30 min up to 270 min, a 500 μL aliquot of the sample was removed. To this aliquot, 3 μL of Tali® Dead Cell Red Reagent was added and then incubated for 1–2 min at room temperature. Three 100 μL aliquots of the stained sample were run on a flow cytometer, and a total of 10 measurements on the Tali® Image Cytometer were made using the remaining sample. Over time, the cells progressed from 95% viable at time 0 to less than 60% viable by 270 min. At every time point, the percentage of the population recorded as viable was comparable on both the Tali® cytometer and the flow cytometer.

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Figure 7. Comparison of apoptosis assays as analyzed on the Tali® Image Cytometer and a flow cytometer. Following a 4-hour induction with camptothecin, Jurkat cells were stained with the Tali® Apoptosis Kit and assessed for apoptosis using the Tali® cytometer and a flow cytometer. The Tali® Apoptosis Kit contains an annexin V–Alexa Fluor® 488 conjugate and PI to differentiate live (annexin V–negative/PI-negative), dead (PI-positive), and apoptotic (annexin V–positive/PI-negative) cells. Using both platforms, 44% of the cell population were measured as live, 44% apoptotic, and 12% dead, confirming that the Tali® image Cytometer provides quantitative data comparable to data collected by flow cytometry.

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Figure 8. Comparison of RFP expression as analyzed on the Tali® Image Cytometer and a flow cytometer. Four cell types (U2OS, 293MSR, HEKn, and CHO-S) were transduced with a plasma membrane–targeted RFP BacMam construct (CellLight® Plasma Membrane-RFP (C10608)) and then analyzed on the Tali® cytometer to determine transduction efficiency and RFP expression. The number of RFP-expressing cells in each experiment was determined using the Tali® Viability Kit – Dead Cell Green on the Tali® Image Cytometer, and these data were compared to data collected from the same samples on a flow cytometer. For each cell type, the percentage of the total population expressing plasma membrane RFP is shown compared with flow cytometry. For most cell types tested, the percentages of RFP-expressing cells detected with the Tali® Image Cytometer were comparable to those obtained using flow cytometry. With the HEKn cell line there was lower correlation between the Tali® cytometer and the flow cytometer, which is caused by the difference in how the Tali® cytometer and a flow cytometer measure clumping cells. However, with both instruments, there was an equivalent drop in the percentage of cells that are considered expressing, when dead cells were removed by gating. [Note: Measuring only the viable cells expressing RFP (by excluding cells that stain with a dead cell marker) provides a more accurate measure of the usable cells in a population.]