Determination of Mammalian Cell Viability on the Tali® Image-Based Cytometer
LIVE/DEAD® Viability/Cytotoxicity Kit
The easy-to-use LIVE/DEAD® Viability/Cytotoxicity Kit allows you to stain cells directly in complete media, with no wash steps required. Analyzing samples with the Tali® Image-Based Cytometer provides fast, accurate quantification of live and dead cells.
Cell viability and vitality determinations are important both for measuring the cytotoxicity of various molecules and drugs, and for verifying the health of cells in culture during propagation. Many assays determine cell viability by using membrane-impermeant reagents to identify dead or dying cells with reduced membrane integrity.
Cells that successfully exclude these reagents are considered viable. In contrast, cell vitality assays directly measure live cells by detecting active metabolic or energetic processes. In this application note, we demonstrate the ability of the Tali® Image-Based Cytometer to quantify both cell viability and vitality quickly and easily when used with the LIVE/DEAD® Viability/Cytotoxicity Kit.
The kit contains two stains—calcein AM and ethidium homodimer-1 (EthD-1)—that measure vitality and viability, respectively. Calcein AM, the vitality dye, is a nonfluorescent, cell-permeant stain that is converted to calcein by ubiquitous intracellular esterases. Calcein is then retained in the cell and becomes brightly fluorescent in the green channel. Only live cells with intact esterase activity are labeled with calcein.
The calcein AM vitality stain is used in conjunction with EthD-1, a membrane-impermeantviability dye that only enters cells with damaged membranes. Once inside the cell, EthD-1 binds to nucleic acids and undergoes a 40-fold enhancement influorescence, making EthD-1–labeled dead cells brightly fluorescent in the red channel.
Materials and Methods
Determination of calcein AM and EthD-1 specificity
Jurkat cells were split into two aliquots of equal cell densities. One aliquot was returned to growth conditions (37°C, 5% CO2), and the second aliquot was treated at 65°C for 1.5 hours to heat-kill the cells. Cells returned to growth conditions were used to represent the live-cell population, and the heat-killed cells were used to represent the dead-cell population. Cells were stained using the LIVE/DEAD® Viability/Cytotoxicity Kit according to the flow cytometry protocol in the product information sheet.
Briefly, cells were labeled with 100 nM calcein AM or 10 μM EthD-1. Cells were incubated at 37°C for 30 minutes, then analyzed immediately with the Tali® instrument using the “GFP + RFP” assay, where“GFP” represents the calcein signal and “RFP” represents the EthD-1 signal. Unlabeled cells were used as a control to determine the fluorescence threshold.
The threshold is indicated by a blue vertical line on the histogram, and can be manually set by the user. In this experiment, the threshold was set just to the right of the peak obtained from analyzing an unlabeled sample. Values to the right of the threshold were counted as positive, and values to the left were counted as negative.
Determination of vitality and viability
In a separate experiment, a 50:50 mixture of live and heat-killed Jurkat cells was stained with 100 nM calcein AM and 8 μM EthD-1 simultaneously. Cells were then analyzed on the Tali® Image-Based Cytometer as described above.
The live Jurkat cells (stained only with calcein AM) had a bright green fluorescence signal. Calcein AM staining resulted in 97% positive cells in the live-cell sample (Figure 1A), whereas in the dead-cell sample, 0% of the cells were positive (Figure 1B). In the dead-cell sample, 100% of the cells were positive for EthD-1, whereas in the live-cell sample, 4% of the cells were positive (Figure 2). The population of dead cells in the live-cell sample most likely represents standard loss of cell viability during propagation and processing. These data confirm that calcein AM and EthD-1 are specific stains for live and dead cells, respectively.
Additionally, there was no spectral overlap from calcein or EthD-1 in neighboring fluorescence channels (data not shown), making the use of these dyes together in a multiplex experiment ideal. When a 50:50 mixture of live and dead cells was stained with both calcein AM and EthD-1, 50% of the cells were determined to be live (positive for calcein), and 45% of the cells were determined to be dead (positive for EthD-1) (Figure 3); 4% of the cells were counted as positive for both calcein and EthD-1. This population most likely represents dying cells that have compromised cell membranes, allowing EthD-1 to enter the cells, but still have residual esterase activity that results in a positive signal for calcein.
The LIVE/DEAD® Viability/Cytotoxicity Kit provides fluorescent stains to specifically label live and dead cells. When used with the Tali® Image-Based Cytometer, this assay provides quick and easy quantification of cell viability in a sample population.
Figure 1. Specificity of calcein AM for live cells. Jurkat cells were left under cell culture conditions (A) or heat-killed (B). Cells were stained with 100 nM calcein AM and then analyzed on the Tali® Image-Based Cytometer. Calcein AM stains only live cells (A), whereas dead cells havea very low fluorescence signal (B).
Figure 2. Specificity of ethidium homodimer-1 (EthD-1) for dead
Figure 3. Discriminating live and dead cells using calcein AM (A) and ethidium homodimer-1 (EthD-1)(B). A 50:50 mixture of live and heatkilled Jurkat cells was stained with both calcein AM (100 nM) and EthD-1 (8 μM). After staining, cells were analyzed on the Tali® Image-Based Cytometer using the “GFP + RFP” assay. Peaks for both calcein and EthD-1 positive cells were well resolved from background fluorescence. The table presents quantification of live and dead cells as the cellular concentration, percentage of cells, and number of cells counted.
|Fluorescence channel||Concentration||Percentage of total cells||Number of cells|
|GFP only (green, calcein positive)||7.04 x 105 cells/mL||50.3%||1,478|
|RFP only (red, EthD-1 positive)||6.32 x 105 cells/mL||45.2%||1,327|
|GFP and RFP (green/ red dual positive)||5.81 x 104 cells/mL||4.1%||122|
|No GFP, no RFP (no signal)||0.52 x 104 cells/mL||0.4%||11|
Figure 3. Discriminating live and dead cells using calcein AM (A) and ethidium homodimer-1 (EthD-1) (B). A 50:50 mixture of live and heatkilled Jurkat cells was stained with both calcein AM (100 nM) and EthD-1 (8 μM). After staining, cells were analyzed on the Tali® Image-Based Cytometer using the “GFP + RFP” assay. Peaks for both calcein and EthD-1 positive cells were well resolved from background fluorescence. The table presents quantification of live and dead cells as the cellular concentration, percentage of cells, and number of cells counted.