Annexin V-FITC vs Propidium Iodide Quantitation of Apoptosis and Necrosis

Apoptosis

Control Samples for Annexin V-FITC vs. Propidium Iodide
Quantitation of Apoptosis and Necrosis.

Christopher L. Reed and Philip F. Mixter
Department of Microbiology, Washington State University, P.O. Box 644233, Pullman, WA 99164-4233

Abstract. Precise cytometric detection and quantitation of cells undergoing programmed cell death (known as apoptosis), cells dying from necrosis, or cells remaining viable within an experimental sample requires consistent and accurate controls. In this article, we relay a protocol for generating these control populations employing the Jurkat human T-cell line. Staining of Jurkat cells treated to either retain viability, induce necrosis or induce apoptosis with fluorchrome-conjugated annexin V and DNA-binding dye propidium iodide delineates these control populations for cytometric analysis. These controls ensure consistent analysis of experimental samples.

Introduction. Cellular proliferation or mitosis is a fundamental biological process. Research over the last decade has identified an equally universal role for cell death. Two basic mechanisms for cell death been elucidated (1). Apoptosis or programmed cell death is distinguished from necrosis because it is highly regulated, requires new gene expression, leads to changes in nuclear morphology, DNA laddering and membrane blebbing (2). The changes in membrane composition lead to extracellular exposure of phosphatidylserine (PS) residues and occur early in the apoptotic cycle, regardless of the initiating signal (3). Exposed PS residues avidly bind annexin V, a natural ligand, in a calcium-dependent manner (4). Membrane changes leading to PS exposure occur rapidly in apoptotic cells, while the cell loses membrane integrity later in the apoptotic process. Necrotic cells expose PS and lose membrane function simultaneously soon after cell injury (4). Using a DNA binding dye such as propidium iodide (PI) in tandem with fluorochrome-conjugated annexin V, apoptotic cells are identified and discriminated from necrotic cells (3).

To meet growing demand, flow cytometry lab facilities must have functional assays to detect and differentiate apoptotic and necrotic cells. These assays must reliably determine if a cell is viable, apoptotic, or necrotic. However, before any new assay is implemented to study a research problem, one must be confident that it yields consistent results. The implementation of effective controls will ensure consistent analysis.

Our laboratory adapted published methods and employed the Jurkat human T cell leukemia cell line to provide these controls. Treating cells with ethanol damages membranes and effectively induces necrosis (2). Apoptosis was induced by ligating the cell surface receptor CD95R (Fas), a member of the tumor necrosis receptor (TNFR) family (6, 7).

Untreated Jurkat cells, Jurkat cells stimulated to undergo apoptosis by CD95R, or Jurkat cells treated with ethanol to induce necrosis provide these control populations. Using these populations as standards, cytometric analysis using the annexin V-PI detection method was completed with confidence.

Results. Detection of apoptosis or necrosis of Jurkat cells.

Jurkat cells were treated with either immobilized anti-CD95R antibody (clone CH11) for 3 hours or 10% (v/v) ethanol for 90 minutes. Untreated cells and treated cells were sequentially stained with annexin V-FITC and PI and then analyzed by flow cytometry. Data plots were generated from analysis of ungated data (Figures 1-3). Apoptotic cells staining with annexin V-FITC, but not PI, appear in the lower right (LR) quadrant of data plots. Necrotic cells appear in the upper right (UR) quadrant, staining with both PI and annexin V-FITC. Untreated Jurkat cells had 3.05% annexin V-FITC binding alone, while 5.02% of the population stained with both annexin V-FITC and PI (Fig. 1). 57% of cells treated with anti-CD95R antibody bound annexin V-FITC (Figure 2), with 51.3% excluding PI. Although Jurkat cells treated with ethanol also showed much higher levels of annexin V-FITC binding than the untreated cells, the pattern of staining was quite different from anti-CD95R antibody-treated cells. Figure 3 shows 42.21% of ethanol-treated Jurkat cells bind both annexin V-FITC and PI. In summary, anti-CD95R antibody treatment induced apoptosis in 51.30% of Jurkat cells, while ethanol treatment induced necrosis in 42.21% of the population.

Figure 1

Figure 2

Figure 3

Discussion. These results demonstrate clear detection of three populations (viable, apoptotic, necrotic) based on treatment. The background was quite low in the untreated cells, approximately 8% of the total population. This single positive and double positive staining of untreated cells (LR and UR quadrants) are most likely cells under going natural senescence. This low background makes it relatively easy to identify large cell populations undergoing apoptosis or necrosis following stimulus or injury.

These control cell populations ensured that the staining protocol was working correctly and can provide single color controls for these types of annexin V-PI analyses. Single color controls were obtained by using CH11-stimulated cells stained only with annexin V-FITC (apoptotic cell populations) or ethanol treated cells stained with only PI (necrotic cell populations). These controls allowed the X and Y means of these populations to be aligned with each other by compensation. We have observed a strong PI signal from FL2 that is detected in the FL1 channel. We have alleviated this problem in certain cell types by using single color controls and adjusting FL2-FL1 compensation.

Materials and Methods.
Culture conditions. Jurkat cells were obtained from the ATCC (TIB-152, Rockville, MD). Jurkat cells were routinely subcultured in complete media [RPMI 1640 media (Cellgro, Herndon, VA) containing 10% (v/v) fetal bovine serum (Life Technologies, Inc. (LTI), Grand Island, NY) in 1mM sodium pyruvate (LTI), 5 x 10 e-5 M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO), 4500g/L glucose (Sigma), 800mg/L glutamine (LTI)] at 3-4 day intervals in a humidified 37°C incubator in 7.5% (v/v) CO2 to maintain log phase growth.

Induction of Apoptosis. Jurkat cells from log phase cultures were counted and assessed for viability by Trypan-Blue (Sigma) exclusion. One million cells per well were dispensed into 6 well culture plates. The cells were centrifuged at 200x g for 5 minutes in order to bring cells into contact with the immobilized antibody. After centrifugation, the plates were incubated for 3 hours at 37°C. Untreated cells were manipulated in the same fashion but dispensed into wells without antibody. Culture plates were coated with antibody by incubation with 2ml/well of 0.1 µg/ml anti-CD95R (clone CH11; MBL, Watertown, MA) in PBS for 48 hours at 4°C. The wells were rinsed once with 4 ml/well cold, sterile phosphate-buffered saline solution before immediately adding cells in culture media.

Induction of Necrosis. Jurkat cells from log phase cultures were counted and assessed for viability by Trypan Blue (Sigma) exclusion. One million cells per well were dispensed into 6 well plates and centrifuged for 5 minutes at 200x g. After centrifugation, ethanol was added to each well to reach a final concentration of 10% (v/v). The cells were incubated for 90 minutes at 37°C.

Annexin V-FITC Propidium Iodide Staining. Cells were transferred from a culture well to a staining tube and washed with 4 ml of 4°C PBS containing 1% (v/v) bovine calf serum (Hyclone, Logan, UT). Cells were centrifuged 10 minutes at 200x g at 4°C and the supernatant removed. 100µl of 2µg/ml annexin V-FITC (Caltag Laboratories, Burlingame, CA) in annexin V-binding buffer [10mM HEPES (LTI), 140mM NaCl, 5mM KCl, 1mM MgCl2, 2.5mM CaCl2, pH 7.4] was added to the cells and the staining tubes were incubated for 10 minutes on ice in the dark. PI single color control cells were treated with 100ul of annexin V-binding buffer alone. After adjusting the total volume of each tube to 0.5ml with annexin V-binding buffer, 1µg per tube of PI (Sigma) was added. The cells were analyzed within 20 minutes by flow cytometry.

Flow Cytometry. All data was acquired with a Becton-Dickinson FACSCalibur single laser cytometer. Although cytometer settings vary, the ranges for cytometer settings during acquisition were: FSC (Voltage e-1, Amp/Gain 3.0, linear scale), SSC (Voltage 347, Amp/Gain 1.0, linear scale), FL1 voltage 450-500 mV, FL2 voltage 450-500 mV, Compensation FL1-(0.8-1.3%)FL2, FL2-(25-30%)FL1, Threshold (FSC at 52). Live statistics were used to align the X and Y mean values of the annexin V-FITC- or PI-stained quadrant populations by compensation. Quadrant markers were assigned using single color control staining samples. Data analysis was performed with either CellQuest (Macintosh platform) or Winmdi (Windows Platform) programs.

References

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