Benchtop GFP/RFP Transfection and Viability Determination Has ArrivedThe Tali™ Image-Based Cytometer is a three-channel benchtop assay platform that uses state-of-the-art optics and sophisticated digital image analysis to perform assays for cells in suspension. With bright-field, green-fluorescence, and red-fluorescence channels, the Tali™ cytometer performs cell counting as well as other common measurements, including GFP/RFP transfection efficiency, GFP/RFP expression, cell viability (live, dead, and total cells), and apoptosis. The Tali™ Image-Based Cytometer is equipped to measure in the green channel (458 nm excitation with a 525/20 nm bandpass emission filter) and in the red channel (530 nm excitation with a 585 nm longpass emission filter). It also provides simultaneous visualization of the cells in bright-field and fluorescence channels, so that data can be visually confirmed.
Three Kits for Monitoring Cell Viability
Currently, three kits have been developed for the Tali™ Image-Based Cytometer. Both the Dead Cell Red Tali™ Viability Kit and the Dead Cell Green Tali™ Viability Kit contain cell-impermeant fluorogenic DNA-binding dyes used for identifying necrotic cells: propidium iodide (PI) and SYTOX® Blue dyes. Monitored in the red channel of the Tali™ instrument, the Dead Cell Red kit is ideal for assessing viability in GFP-expressing cells; Dead Cell Green reagent is perfect for determining cell viability in RFP-expressing cells. For apoptosis analyses, the Tali™ Apoptosis Kit–Annexin V Alexa Fluor® 488 and Propidium Iodide is also available.
Quantitative Data in Minutes
The quantitative measure of cell viability is an important consideration for any laboratory performing cell culture and/or cellular assays. The Tali™ Image-Based Cytometer and Tali™ Viability kits enable researchers to get reliable, quantitative cell viability data in minutes.
In Figure 1, the Tali™ Image-Based Cytometer was used to measure the fluorescent protein expression level and cell viability in cells that were transduced with BacMam 2.0–based CellLight® Nucleus-GFP.
In Figure 2, four representative cell types were transduced with CellLight® Nucleus-GFP, and the number of cells expressing GFP was quantified using the Tali™ cytometer and compared with data from the same samples analyzed on a flow cytometer. After staining with the Tali™ Viability Kit–Dead Cell Red, the dead cell population was identified on the Tali™ instrument and on the flow cytometer. For each of the four cell lines tested, quantitative cell viability and GFP expression data obtained using the Tali™ Image-Based Cytometer were comparable to data obtained using a flow cytometer. Similar results were obtained for cells that were transduced with CellLight® Plasma Membrane-RFP (data not shown; see Measuring RFP expression in viable cells using the Tali™ Image-Based Cytometer for additional details).
Figure 1. Assessment of GFP-expressing cells and viability with the Tali™ Image-Based Cytometer. 293MSR cells transduced with CellLight® Nucleus-GFP were analyzed on the Tali™ instrument. The histograms in panels (A) and (B) show the GFP and PI fluorescence profiles, respectively, for the transduced populations. As the user adjusts the thresholds for these fluorescence assignments, the visual display updates to reflect the cells that meet the new threshold requirements. (C) Colored circles can be viewed on the image for easy identification of cells that were counted in a given subset of the population. Colored circles represent: live, GFP-expressing cells (green circles); live, non–GFP-expressing cells (blue circles); dead, GFP-expressing cells (yellow circles); and objects discounted by cell size gating (black circles).
Figure 2. Comparable measurements of GFP expression and viability on both the Tali™ Image-Based Cytometer and a flow cytometer. Cells were transduced with a GFP expression construct and assayed using the Tali™ Viability Kit–Dead Cell Red. The percentage of the population expressing GFP (GFP) and the percentage that is both viable and expressing GFP (+ viable) are shown.
Use of Dead Cell Red for Viability Studies
In addition to offering a dead-cell stain compatible with GFP-expressing cells, the Tali™ Viability Kit–Dead Cell Red can be used to monitor cell viability in response to environmental stimuli. To demonstrate this, five cell types, including commonly used cell lines and primary cells, were assayed using the Tali™ Viability Kit–Dead Cell Red reagents and protocols. The viability data obtained were shown to be in agreement with results obtained independently using a flow cytometer (Figure 3).
In a time course assay (Figure 4), 10% (v/v) ethanol was added to 293MSR cells to induce cell death. Cells were removed every 30 minutes over a period of 270 minutes, and Tali™ Dead Cell Red reagent was added. Three 100 μL aliquots of the stained sample were run on a flow cytometer, and a total of 10 measurements on the Tali™ Image-Based Cytometer were made using the remaining sample. Again the viability data generated using the Tali™ instrument were in agreement with results from a flow cytometer (Figure 4).
Figure 3. Comparable results for viability measurements using the Tali™ Image-Based Cytometer and a flow cytometer. Percent viable cells as assessed using the Tali™ Viability Kit–Dead Cell Red is shown in five different cell types. Percent viable cells detected using the Tali™ Image-Based Cytometer (grey bars) or a flow cytometer (green bars) is indicated.
Figure 4. Time course comparison of cell viability as analyzed with the Tali™ Image-Based Cytometer and a flow cytometer. Population data measured with the Tali™ Image-Based Cytometer are comparable to those obtained using a flow cytometer. Over time, the population 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 the same on both the Tali™ Image-Based Cytometer and the flow cytometer.
Determination of GFP and RFP Co-Expression On the Tali™ Instrument and a Flow Cytometer
The Tali™ Image-Based Cytometer can also be used to measure transduction efficiencies in cells that are co-transduced with nuclear-targeted GFP and plasma membrane–targeted RFP BacMam 2.0 expression constructs.
In one study involving transduction of four different cell types, the numbers of cells that were expressing only GFP, expressing only RFP, or coexpressing GFP and RFP were reported by the Tali™ instrument and compared with data from the same samples run on a flow cytometer. For each transduced cell line, the percentage of GFP- and/or RFP-expressing cells reported by the Tali™ instrument was comparable to measurements from the flow cytometer (see Measuring GFP/RFP expression using the Tali™ Image-Based Cytometer).
Cell Viability and Vitality in One Powerful Package
The small yet powerful Tali™ Image-Based Cytometer offers two-color fluorescent quantitative analysis for routine endpoint assays, such as cell viability and two-color apoptosis/vitality assays, that is not possible with a quick microscope check. In addition, it is the ideal companion instrument for flow cytometry and GFP/RFP transfection workflows, for confirming critical parameters before setting up more complicated flow cytometer experiments.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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