Quantitating GFP & 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. In addition, 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 researchers with 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.
In this application note, we demonstrate that the Tali® cytometer delivers accurate cell counting and GFP and RFP fluorescence data for four BacMam transduced cell lines. Calculated concentration and population percentage are reported for GFP expression and RFP expression of the sample. These data are in concordance with the results obtained using flow cytometry.
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). The channels in the Tali® cytometer were optimized for emeraldGFP and tagRFP protein expression experiments.
U2OS, 293MSR, HEKn, and CHO-S cells were transduced using a nucleartargeted GFP (CellLight® Nucleus-GFP) and 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 of 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 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 levels in cells that were co-transduced with nuclear-targeted GFP and plasma membrane-targeted RFP BacMam 2.0 expression constructs. For the four cell types transduced, the number of cells that were expressing only GFP, only RFP, or co-expressing GFP and RFP was reported by the Tali® instrument and compared with data from the same samples run on a flow cytometer.
To exclude debris from the sample being analyzed, the cell size gate on the Tali® 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 (thresholding) on the histograms generated from the cell data provided 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 within a given 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 nonexpressing 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 slightly shifted. 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- and RFP-expressing cells with the Tali® Image Cytometer. U2OS cells transduced with CellLight™ Nucleus-GFP and CellLight™ Plasma Membrane-RFP BacMam constructs were analyzed on the Tali® instrument. The histograms in panels A and B show the red and green 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: cells expressing GFP (green circles), cells expressing RFP (red circles), cells expressing both GFP and RFP (yellow circles), and objects discounted by cell size gating (black circles).|
Accurately quantitating the cellular transduction efficiency of GFP and RFP constructs can be extremely challenging to today’s life science researcher. The Tali® Image Cytometer can identify and quantitate populations of cells that are differentially expressing GFP and RFP. The graphical representation in Figure 2 shows the percent of the total population for each cell type that is expressing GFP and RFP. When co-transduced with both GFP and RFP, three of the cell types showed a high percentage of cells expressing both proteins. It is interesting to note that the HEKn cells (human epidermal keratinocytes, neonatal) have a higher percentage of cells expressing the GFP construct than the other cell types, and a lower percentage of cells positive for both GFP and RFP. These data demonstrate that the Tali® cytometer can distinguish population of cells expressing GFP and RFP in both green and red channels.
|Figure 2. Assessment of GFP and RFP transduction in four different cell lines. The transduction efficiency of each cell type with these particular constructs is unique and demonstrates the ability of the Tali® cytometer to measure varying levels of fluorescence in two channels. Percentage of the cell populations expressing GFP and/or RFP is indicated in the graph: GFP-positive, RFP-negative cells (grey bars), GFP-negative, RFP-positive cells (green bars), and GFP-positive, RFP-positive cells (purple bars).|
The Tali® cytometer can measure GFP and RFP accurately. A comparison of the percent expression recorded by the Tali® cytometer as compared to the flow cytometer is shown in Table 1. The transduction efficiency for each of these four cell types is at least 35%, and population statistics for each combination of fluorescent protein matched well between the Tali® cytometer and a flow cytometer.
Table 1. Comparison of the assessment of GFP/RFP cell populations between the Tali Image-Based Cytometer and a standard flow cytometer.
For each transduced cell line, the Tali® Image Cytometer produced GFP and RFP expression data comparable to the results obtained by a flow cytometer (Table 1), but in a fraction of the time. In addition to producing quantitative expression data, the Tali® cytometer allows the calculation of transduction efficiency for each of the constructs individually and for the co-transduced cells.
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).
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.
|Figure 3. The Tali® Image Cytometer GFP+RFP display after measuring U2OS cells transduced with CellLight™ Nucleus-GFP and CellLight™ Plasma Membrane-RFP BacMam. 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.|