Identifying Cell Viability With the Tali® Image Cytometer
Propidium iodide (PI) is a fluorogenic DNA-binding dye that can pass through the compromised membranes of dead cells. Using the Tali® Viability Kit – Dead Cell Red (containing PI) with the Tali® Image Cytometer, the number of live and dead cells in a sample of up to 5,000 cells can be determined. The use of this assay allows a quick assessment of the population’s health and/ or response to environmental stimuli. Here, we demonstrate that the Tali® Image Cytometer delivers accurate cell counts for total cells, live cells, and dead cells.
GFP/RFP Transfection & Cell Viability on Tali
The quantitative measure of cell viability is an important consideration for any laboratory performing cell culture and/or cellular assays. Viability studies are often used as part of routine screening of cell lines and for assessing the cytotoxicity of environmental factors. A common technique for determining the viability of a population of cells is to perform a dye-exclusion assay. In this type of assay, dead cells are stained with a small-molecule dye that can only enter cells with compromised plasma membranes. In healthy cells, the uncompromised cytoplasmic membrane excludes the dye, leaving live cells unstained. To measure viability in large populations of cells, however, researchers have the painstaking task of counting thousands of cells by eye or they must purchase expensive instrumentation to quickly and accurately quantitate the number of live and dead cells. The Tali® Image Cytometer is a small yet powerful benchtop device that solves this dilemma, providing researchers with reliable, quantitative viability data in minutes.
The five cell types in this study, including commonly used cell lines and primary cells, were assayed using the reagents and protocols from the Tali® Viability Kit – Dead Cell Red. Viability data obtained from the Tali® Image Cytometer were shown to be in agreement with results independently obtained using a flow cytometer, demonstrating accurate, quantitative data from a convenient benchtop device.
Materials & Methods
The Tali® Image Cytometer is capable of measuring cellular fluorescence that falls within the two fluorescent channels of the instrument: (1) 458 nm excitation with a 525/20 nm emission filter (green channel); and (2) 530 nm excitation with a 585 nm longpass emission filter (red channel). The fluorescent dye in the Tali® Viability Kit was matched to the red channel of the Tali® cytometer, making it perfect for viability analysis alone or in combination with GFP-expressing cells or other common green fluorophores such as FITC and Alexa Fluor® 488. The assay uses minimal cell sample and reagent and can be performed in cell growth medium typically in less than 5 minutes.
All cells were grown in T75 flasks using appropriate media with or without serum until they reached approximately 1 x 106 cells/mL. Jurkat cells were transferred directly from the flask into a 50 mL conical vial. All adherent cell lines were harvested using TrypLE™ reagent, counted using the Countess® Automated Cell Counter, and then resuspended in their growth medium to a final concentration of 1 x 106 cells/mL.
To compare the accuracy of viability counts measured with the Tali® cytometer and a flow cytometer, cells were divided into 100 μL samples, and 1 μL of Tali® Dead Cell Red reagent was added. Following a 1–2 minute incubation, cells were analyzed on the Tali® Image Cytometer and 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, cells were collected every 30 minutes over a period of 270 minutes and incubated with Tali® Dead Cell Red reagent. Three 100 mL 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.
Results and Discussion
To compare the accuracy of the viability data from the Tali® Image Cytometer with data obtained with a flow cytometer, 293MSR cells were exposed to 10% ethanol to induce cell death at various time points. At each time point, cells were stained using the Tali® Viability Kit and assayed on the Tali® Image Cytometer and on a flow cytometer. To exclude debris from the samples being analyzed, the cell size gate on the Tali® cytometer was used, allowing the instrument to include only the cells of interest in downstream fluorescence analysis.
Stained cells were separated from autofluorescent cells by setting a minimum fluorescence value (threshold) on the histograms generated by the Tali® cytometer. The fluorescence thresholds were then visually confirmed using the cell image overlays of bright-field in the red fluorescence channel with circles, which indicated how each individual cell was categorized by fluorescence. By setting the threshold just to the right of the dimmest peak, cells to the left of the threshold were excluded from those counted as PI-positive in the red channel (Figure 1A and 1B). An alternative way to identify autofluorescent cells is to measure a sample of cells that is not stained with PI. The peak in the fluorescence histogram of non-expressing cells represents cellular autofluorescence, and the threshold can be set just to the right of this peak for subsequent runs on an individual day. It should be noted, however, that in samples where the positive fluorescence is bright, the autofluorescence peak may be shifted slightly to the right. In all cases, the fluorescence threshold setting was confirmed visually in the image, confirming that the reported data were derived accurately (Figures 1C–F).
|Figure 1. Assessment of cell viability with the Tali® Image Cytometer. 293MSR cells stained with the Tali® Viability Kit were analyzed on the Tali® instrument at 0 (A, C, E) or 270 (B, D, F) minutes after induction with ethanol. The histograms in (A) and (B) show the PI fluorescence profile for the cell populations. As the user adjusts the thresholds for these fluorescence assignments, the visual display (C and D) is updated to reflect those cells in the population that meet the threshold requirements. Colored circles can be viewed on the image to allow easy identification of cells that were counted in a given subset of the population (E and F); colored circles are designated: live, PI-negative cells (blue circles), dead, PI-positive cells (red circles), objects discounted by cell size gating (black circles).|
To determine if comparable cell viability data could be collected on the Tali® instrument and a flow cytometer in multiple cell types, Jurkat, U2OS, 293MSR, HEKn, and CHO-S cells were stained with the Tali® Viability Kit and analyzed on the Tali® cytometer and a flow cytometer. Percentages of the population reported as live by both the Tali® cytometer and the flow cytometer were compared. In common cell lines (JKT, U2OS, 293MSR, and CHO-S), viability was measured typically in the range of 90 to 100% viable with both the Tali® Image Cytometer and the flow cytometer, while the primary cell cultures (HEKn) showed a somewhat lower viability of 67% (Figure 2).
|Figure 2. Comparison of viability in cell populations between the Tali® Image Cytometer and a flow cytometer. Percent viable cells of the total population as assessed by the Tali® Viability Kit is shown in five different cell types. Percent viable cells detected by the Tali® Image-Based Cytometer (grey bars) or flow cytometer (green bars) is indicated.|
To compare viability results between the Tali® cytometer and a flow cytometer during a time course, 293MSR cells were collected at various time points following ethanol induction and stained with the Tali® Viability Kit. Cell viability was assessed at each time point on the Tali® cytometer and a flow cytometer. An increase in cell death (96% to 58% viable), as indicated by an increase in PI staining, was detected over the 4.5 hour period. For each time point, low variability was observed between results recorded on two different Tali® Image Cytometers, and two data sets matched well with the mean viability measurements from the flow cytometer. Note that the increased variability within replicates at each time point for the flow cytometry data is attributed to the additional time needed to prepare the cells and instrument for analysis at each time point (Figure 3). For each cell line and time point tested, viability results from the Tali® cytometer were consistent with those obtained with the flow cytometer.
|Figure 3. Time course comparison of cell viability as analyzed with the Tali® Image Cytometer and a flow cytometer. Population data measured with the Tali® Image Cytometer are comparable to those obtained using a flow cytometer. Over time, the cells progressed from 95% viable at time 0 to less than 60% viable by 270 minutes. At every time point, the percent of the population recorded as viable was the same on both the Tali® Image Cytometer and the flow cytometer.|
Using the Tali® Viability Kit to determine cell viability, the Tali® Image Cytometer produced quantitative data on live and dead cell populations that were comparable to results obtained with a flow cytometer. The speed and ease of use of the Tali® instrument allows researchers to quickly and accurately measure the concentration and health of cells without leaving their bench, and to obtain quantitative information on the progress of cell death brought about by environmental factors. The Tali® cytometer allows simultaneous visualization of cells in bright-field and fluorescence channels. Because the display updates after adjusting threshold settings for the counting and fluorescence algorithms (Figure 4), higher confidence in data accuracy is obtained. This small yet powerful instrument offers quantitative analysis for routine end-point assays such as cell viability and two-color apoptosis/vitality assays. In addition, it is the ideal companion instrument for flow cytometry workflows, allowing for the confirmation of critical parameters before setting up more complicated flow cytometer runs.
|Figure 4. The Tali® Image Cytometer viability display after measuring U2OS cells stained with the Tali® Dead Cell Red dye. The assignment of each cell with a particular fluorescence channel is dependent on the threshold set in the fluorescence histogram associated with that channel. After resetting the threshold, the circles in the image on the left side of the screen will update to reflect the new fluorescence levels.|