Measuring Apoptosis With the Tali® Image Cytometer
Apoptosis is a widely studied cell death process that plays an important role in the biology of cancers and tumors. Many cell biology researchers need an inexpensive, easy-to-use assay platform that quickly, thoroughly, and accurately analyzes apoptotic parameters. The Tali® Image Cytometer provides researchers with a rapid and comprehensive platform to assess thousands of cells in a population, right from their benchtop. Using the Tali® Apoptosis Kit with annexin V–Alexa Fluor® 488 and propidium iodide and the Tali® Image Cytometer, the numbers of live, dead, and apoptotic cells can be determined, allowing a rapid and quantitative assessment of the effects of environmental stimuli on apoptosis.
In normal live cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. In apoptotic cells, PS is translocated from the inner to the outer surface of the plasma membrane. In leukocyte apoptosis, PS residing on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages. The human anticoagulant annexin V is a 35–36 kDa Ca2+–dependent phospholipid-binding protein that has a high affinity for PS. An annexin V conjugate labeled with a fluorophore such as Alexa Fluor® 488 can be used to identify apoptotic cells by binding to the exposed PS.
The Tali® Apoptosis Kit contains both a green-fluorescent annexin V–Alexa Fluor® 488 conjugate to identify apoptotic cells and red-fluorescent propidium iodide (PI) to identify dead or necrotic cells. Cell samples stained with these reagents can be divided into three populations—apoptotic cells (green fluorescence), dead cells (red fluorescence or yellow fluorescence resulting from a combination of red and green fluorescence), and live cells (little to no fluorescence) (Figure 1). The Tali® Image Cytometer captures up to 20 images (i.e., fields of view) of the stained sample, automatically analyzes the images with sophisticated digital image–based cell counting and fluorescence detection algorithms, and displays the results of the analysis in the Measure tab (Figure 2). The data from the analysis, including the image files, can be immediately downloaded to a USB drive and transferred to a computer for sample comparisons.
|Figure 1. Results obtained from the Tali® Apoptosis Assay. The Tali® Apoptosis Assay uses annexin V and PI to assess cell viability and health. Typical results indicate how each cell is analyzed (live, dead, and apoptotic).|
|Figure 2. The Tali® Image Cytometer apoptosis display after measuring Jurkat cells stained with the Tali® Apoptosis Kit. 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 Tali® Image Cytometer is a valuable tool for routine cell analysis, delivering quantitative data unavailable from microscopic analysis. 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.
Here, we demonstrate that the Tali® Image Cytometer delivers accurate quantitative analysis of live, dead, and apoptotic cell populations for the Jurkat cell line using the Tali® Apoptosis Kit.
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 fluorescent dyes in the Tali® Apoptosis Kit—Annexin V–Alexa Fluor® 488 conjugate and propidium iodide (PI), were matched to the channels of the Tali® cytometer, and the assay was optimized for a streamlined workflow. The entire assay, including washes, staining, and measurement on the instrument, typically takes 30 minutes or less.
Jurkat cells were grown in a T75 flask using RPMI with 10% serum until they reached approximately 1 x 106 cells/mL. Camptothecin was added directly to the cells to a final concentration of 10 μM, and the sample was incubated for 4 hours at 37°C, 5% CO2. The concentration of cells was measured by the Countess® Automated Cell Counter. Cells were harvested by trypsin treatment using TrypLE™ reagent and stained using the Tali® Apoptosis Kit. The sample was divided and analyzed independently on both the Tali® Image Cytometer and a flow cytometer following the manufacturer’s recommended protocol.
Results & Discussion
Apoptotic induction was analyzed in Jurkat cells 4 hours after treatment with camptothecin using the Tali® Image Cytometer. To determine the portion of the population that had become apoptotic, cells were stained with the annexin V–Alexa Fluor® 488 conjugate. Propidium iodide (PI) was used to differentiate the cells that were dead (annexin V positive/PI positive or annexin V negative/PI positive) from those that were apoptotic (annexin V positive/PI negative). The percentages of the population reported as viable, apoptotic, and dead by the Tali® cytometer were comparable with data from the same samples independently run on a flow cytometer.
In order 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 (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 3a and 3b). An alternative way to identify autofluorescent cells is to measure a sample of cells that is not stained with a dye. The peak in the fluorescence histogram of unstained 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 confirmed visually in the image, which confirmed the reported data was being derived accurately (Figures 3c and 3d).
|Figure 3. The Tali® cytometer allows confirmation of cell and fluorescence assignments using the visual display of the sample. Jurkat cells induced with 10 μM camptothecin were analyzed on the Tali® instrument. The histograms show the Alexa Fluor® 488 (A) and propidium iodide (PI) (B) fluorescence profiles for the induced populations. As the user adjusts the thresholds for these fluorescence assignments, the visual display (C) is updated to reflect those cells in the population that meet the threshold requirements. After analysis, colored circles can be viewed on the image to allow easy identification of cells that were counted in a given population (D); colored circles are designated: live cells, annexin V–Alexa Fluor® 488 negative/PI negative (blue circles); apoptotic cells, annexin V–Alexa Fluor® 488 positive/PI negative (green circles); dead cells, annexin V–Alexa Fluor® 488 positive/PI positive (yellow circles); dead cells, annexin V–Alexa Fluor® 488 negative/PI positive (red circles); and objects discounted by cell size gating (black circles).|
The Tali® Image Cytometer was able to accurately measure cells in the population stained with the annexin V–Alexa Fluor® 488 conjugate and/or PI. The average of ten measurements on the Tali® cytometer and six measurements on the flow cytometer are displayed in Figure 4. Both the Tali® cytometer and the flow cytometer report that after 4 hours of induction with camptothecin, 44% of the Jurkat cell population is apoptotic, 44% are viable, and 12% are dead. As these data demonstrate, the population statistics obtained from the Tali® cytometer and flow cytometer are in agreement.
|Figure 4. Comparison of apoptosis analysis between the Tali® Image Cytometer and a flow cytometer. Following a 4 hour induction with camptothecin, Jurkat cell populations were assessed for apoptosis using the Tali® cytometer and a flow cytometer. Using both platforms, 44% of the cell population were live, 44% were apoptotic, and 12% were dead, confirming the Tali® Image Cytometer provides quantitative data comparable to data collected by flow cytometry.|
For the Jurkat model of apoptosis, the Tali® cytometer produced live, dead, and apoptotic data comparable to the results given by the flow cytometer (Figure 4), but in a fraction of the time. The speed and ease of use of the Tali® instrument allow researchers to quickly measure multiple time points in the apoptotic pathway without leaving their bench. 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 higher 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 experiments.