Measuring Viability in RFP Expressing Cells With the Tali® Image Cytometer
The Tali® Image Cytometer is a valuable tool for routine cell analysis, delivering quantitative data unavailable from a quick microscope check. The Tali® cytometer allows accurate analysis for many of the routine cell health and vitality protocols that are cumbersome and time consuming by flow cytometry. The instrument also provides a rapid, convenient tool for routine two-color analysis that can be used in place of flow cytometry or as a pre-run verification. With a small footprint, the Tali® Image Cytometer performs several common assays right at the benchtop.
Materials and Methods
The Tali® Image Cytometer is capable of measuring cellular fluorescence that falls within the two fluorescence 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). To accurately assess the viability of RFP-expressing cells, the fluorescent dye in the Tali® Viability Kit – Dead Cell Green (containing SYTOX® Blue) was matched to the green channel in the Tali® instrument. The assay, optimized for a streamlined workflow, uses minimal cell sample and reagent and can be performed in cell growth medium typically in less than 5 minutes.
U2OS, 293MSR, HEKn, and CHO-S cells were transduced using a plasma membrane–targeted RFP BacMam 2.0 expression construct (CellLight® Plasma Membrane-RFP). Briefly, 7.5 x 105 cells were added to a T75 flask along with 0.5 mL prepared BacMam reagent and 9 mL of the appropriate medium. The flask was incubated overnight at 37°C, 5% CO2. The cells were harvested using TrypLE™ reagent and the cell concentration was adjusted to 1 x 106. The sample was divided, stained using the Tali® Viability Kit – Dead Cell Green, and analyzed both on a flow cytometer and on the Tali® Image Cytometer following the manufacturer’s recommended protocol.
Results and Discussion
The Tali® Image Cytometer was used to measure the fluorescent protein expression level in cells that were transduced with a plasma membrane–targeted RFP BacMam 2.0 expression construct (CellLight® Plasma Membrane-RFP). Four cell types were transduced (U2OS, 293MSR, HEKn, and CHO-S) to demonstrate the utility of the instrument. From these transduction experiments, the numbers of RFP-expressing cells were determined by using the Tali® cytometer—these data were compared to data from the same samples collected on a flow cytometer. Additionally, in order to identify the dead population on both the Tali® cytometer and the flow cytometer, cells were stained with the Tali® Viability Kit – Dead Cell Green. In this part of the experiment, the number of live cells expressing RFP in the population was identified.
In order to exclude debris from the sample being analyzed, the cell size gate on the Tali® Image Cytometer was used, allowing the instrument to include only the cells of interest in the downstream fluorescence analysis. Fluorescent cells were separated from autofluorescent cells by setting a minimum fluorescence value (threshold) on the histograms generated from the cell data by the Tali® cytometer. The fluorescence thresholds were then visually confirmed using the cell image overlays of bright-field in each 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 fluorescent in that particular channel (Figures 1A and 1B). An alternative way to identify autofluorescent cells is to measure a sample of cells that is not expressing the fluorescent protein of interest. 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 to the right somewhat. In all cases, the fluorescence threshold setting was checked visually in the representative image, confirming the reported data was being derived accurately (Figures 1C and 1D).
|Figure 1. Assessment of GFP-expressing cells and viability with the Tali® Image Cytometer. 293MSR cells transduced with CellLight® Plasma Membrane-RFP BacMam constructs were analyzed on the Tali® instrument. The histograms in panels A and B show the RFP and SYTOX® Blue fluorescence profiles for the transduced populations. As the user adjusts the thresholds for these fluorescence assignments, the visual display (panel C) 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 population (panel D). Colored circles are designated: dead, non-RFP-expressing cells (green circles); live, non-RFP-expressing cells (blue circles); dead, RFP-expressing cells (yellow circles); live, RFP-expressing cells (red circles); and objects discounted by cell size gating (black circles).|
The addition of Dead Cell Green dye (SYTOX® Blue) gives more accurate data by removing dead cells from the results, thereby measuring only viable cells expressing RFP within the population. The Tali® cytometer was able to accurately measure cells that were both viable and expressing RFP. This was accomplished using the Tali® Viability Kit – Dead Cell Green and setting thresholds on the SYTOX® Blue Dead Cell Stain channel to exclude the dead cells. The graphical representation in Figure 2 shows the percent of the total population for each cell type expressing RFP, side by side with the percent of the total population that is both viable and expressing RFP. By including the Dead Cell Green dye, the final population of the cells that is usable for downstream processing is more obvious. It is important to note that the dead cells in the population could come from various sources, including normal cell death in culture or cell death caused by environmental conditions (i.e., trypsinization or death induced by the transfection/transduction method chosen). These data demonstrate that the Tali® cytometer is capable of measuring the differential between total RFP expression in a population and RFP expression in the live population (Figure 2).
|Figure 2. Comparison of RFP expression and viability in cell populations between the Tali® Image Cytometer and a flow cytometer. Percent of the total population expressing plasma membrane–targeted RFP is shown followed by the percent of the total population that is both viable and expressing. Percentage of RFP-positive cells detected by the Tali® Image Cytometer (grey bars) or flow cytometer (green bars) are indicated.|
For each cell line, the Tali® Image Cytometer produced quantitative RFP expression data, which is not possible with a visual inspection of the cells using a standard fluorescence microscope. The accuracy of these results are comparable to that obtained on a flow cytometer (Figure 2), but results are collected in a fraction of the time. In addition to producing quantitative expression data, a single Tali® cytometer experiment allows the calculation of more than one parameter—in this case, transduction efficiency data was obtained for both the total and viable cell populations.
The Tali® cytometer provides a bright-field image on the instrument display, allowing simultaneous visualization of the cells in bright-field and fluorescence channels. In addition, the display updates after adjusting the threshold settings for the counting and fluorescence algorithms, resulting in more confidence in the accuracy of the final data generated (Figure 3).
|Figure 3. The Tali® Image Cytometer RFP+Viability display after measuring U2OS cells transduced with CellLight™ Plasma Membrane-RFP BacMam and stained with Tali® Dead Cell Green 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.|
The small yet powerful Tali® Image Cytometer 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 confirmation of critical parameters before setting up more complicated flow cytometer runs.