ARTICLE |
Correspondence to: Rita Gatti, Inst. of Histology and General Embryology, University of Parma, Via Volturno, 39, 43100 Parma, Italy.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate. (J Histochem Cytochem 46:895900, 1998)
Key Words: apoptosis, vital dyes, calcein-AM, annexin V-FITC, cell adhesion, confocal laser microscopy, PC 12 cells, NIH3T3 cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Annexin V-FITC is widely employed in cytometry and microscopy as an early marker for apoptosis because of its binding affinity for phosphatidilserine (PS), which is exposed at the cell surface early in the process (
Microscopic evaluation of apoptosis, although somewhat cumbersome, remains the most reliable system of detection. Although many probes are available for DNA staining in confocal microscopy (
Calcein-AM proved to be both specific and sensitive for detection and tracking of apoptosis in living cells by confocal laser microscopy (
In this study we compared the performance of annexin V-FITC and calcein-AM as early apoptosis probes in confocal microscopy. Two different cell lines, PC12 and NIH3T3, were studied in a self-built flow chamber that allows long-term observation of living adherent cells under controlled microenvironmental conditions and repeated culture medium changes without modification of a preset observation field (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Cultures and Treatment
Rat pheochromocytoma PC 12 cells (2 x 104 cells/cm2) were grown on coverslips coated with poly-D-lysine to ensure cell adhesion and were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum. At 24 hours after seeding, coverslips were placed in a special self-built flow chamber, described in detail elsewhere (
Mouse NIH3T3 fibroblast cells (2 x 104 cells/cm2) were grown on coverslips in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. For apoptosis induction (
All reagents for cell culture were purchased from Life Technologies (Gaithersburg, MD). All other chemicals were supplied by Sigma Chemical (Milan, Italy).
Confocal Microscopy
Our images were acquired with a Multiprobe 2001 Molecular Dynamics confocal laser scanning microscope (CLSM) (Sunnyvale, CA) based on a Nikon diaphot inverted microscope and equipped with an argon laser. We employed x40 NA 0.95 dry and x100 NA 0.75 oil immersion Planapo lenses coupled to a 100-µm pinhole according to the cell type under investigation. Image processing was performed on a Silicon Graphics Personal Iris workstation (Image Space Software; Molecular Dynamics).
As stated above, our flow chamber allows repeated medium substitutions with stability of the observation field both laterally (xy-axis) and vertically (z-axis).
Once plated in the flow chamber, cells maintained in normal growth medium do not show morphological signs of distress, adhesion defects, or reduction of cell number up to 24 hr of brightfield observation.
Probe Loading
After 4 hr of staurosporine and 12 hr of L-asparaginase treatment for PC12 and NIH3T3 cells, respectively, the incubation media were replaced with the addition of 1 µg/ml annexin V-FITC and 2 µg/ml propidium iodide. Annexin V-FITC solution (20 µg/ml in Tris-NaCl buffer) was taken from a commercial kit (ApoAlert; Clontech, Palo Alto, CA) and diluted to the suggested working concentration in complete medium.
After 10 min of loading an observation area was chosen in brightfield and section series was acquired with z-step set at 1 µm.
Samples were excited with the 488-nm laser line of CLSM and the emission recorded through a 510-nm primary beamsplitter and a 530 nm DF barrier filter. To assess the presence of propidium iodide in the nuclei, emission over 560 nm was also always checked.
After series acquisition, calcein acetoxymethylester (calcein-AM; Molecular Probes, Eugene, OR) was added to the culture medium in the flow chamber at a final concentration of 2 µM. The dye was taken from a stock solution (1 mM) in dimethylsulfoxide (DMSO). After 10 min of loading, the same field was scanned again with the same filter setting. The signal- to-noise ratio is very high and therefore confocal image acquisition is possible at very low laser power (well below 1 mW).
In another experimental setting, NIH3T3 cells were treated for 12 hr in L-asparaginase, harvested by trypsinization, stained in suspension with annexin V-FITC and propidium iodide, and eventually re-seeded onto a coverslip. After evaluation of positivity for annexin V under CLSM, calcein-AM was added to the culture as previously described.
In all experiments, PC12 and NIH3T3 cells incubated in the absence of apoptogenic stimuli were dye-loaded and used as controls. No toxic effects were observed under these conditions at the experimental times. For each cell type, experiments were replicated three times with comparable results.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PC12 Cells
|
According to our experimental protocol, we added calcein-AM to the culture medium and the entire cell population in the field became appreciable within 10 min (Figure 1B). A heterogeneous pattern could be observed, corresponding to the asynchronous progression of the population through the process. Only a few cells show normal probe distribution, and many elements have intense nuclear fluorescence due to chromatin condensation. Cells that have further progressed present a variable amount of blebs containing highly condensed chromatin. The membrane integrity of the cell remnants is preserved, as can be inferred by the persistence of calcein signal in cytoplasm.
To compare the behavior of the two probes, we have merged the annexin V-FITC and the calcein images (Figure 1C). The latter was evenly attenuated to 60% of the original intensity, because the exceedingly higher efficiency of calcein would overwhelm the weaker signal from annexin V-FITC and render it unappreciable. It is evident that annexin V-FITC labeled only the cells in the early phases (i.e., chromatin condensation, initial blebbing), whereas other cells that have further proceeded (i.e., intense blebbing) are annexin V-FITC negative, as are apparently normal cells.
NIH3T3 Cells
We have previously shown that L-asparaginase induces apoptosis in NIH3T3 cells (
|
|
In cells harvested by trypsin digestion after 12 hr of treatment with L-asparaginase and labeled in suspension, we observed several cells positive for annexin V-FITC (Figure 4A). Calcein addition to the culture reveals many other cells with variable degrees of nuclear condensation (Figure 4B). Annexin-positive cells are calcein-negative but are intensely stained by propidium iodide (Figure 4C), thus indicating that membrane integrity is severely damaged. Consistently, it can be noted, at higher magnification that annexin V-FITC has tagged PS on the inner side of plasma membrane (not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Morphological evaluation of apoptosis is still the less biased procedure available; it should therefore precede any quantitative assessment of the process in previously untested cells.
Annexin V-FITC is presently employed, both in cytometry and microscopy, as an early marker for apoptosis. This method has been proven reliable in suspended cells, whereas its efficiency in adherent cultures is uncertain and only one report is available on this specific matter (
Calcein-AM provides morphological evidence of chromatin condensation and segregation in blebs, together with functional information about plasma membrane competence (
Employing confocal microscopy, we have compared these two different morphological approaches in two cell lines with different adhesion features: PC12 and NIH3T3. The former are not intrinsically adherent and also, on poly D-lysine-coated coverslips, tend to maintain a globular shape and a small adhesion surface. In contrast, the latter adhere spontaneously to the coverslip with a very flat profile and a wide contact surface.
As expected, a significant percentage of PC 12 cells exhibit both PS exposure and condensed chromatin after 4 hr of incubation with staurosporine. Rather surprisingly, however, other cells have further progressed through the apoptotic process, as demonstrated by calcein pattern, but are negative for annexin staining. It appears unlikely that phospholipid asymmetry, if lost at the very beginning of the process, could be restored later, because of the depletion of energetic resources occurring in the late stages of apoptosis (
The same experiment carried out on NIH3T3 cells confirms that at any given time annexin V does not detect the entire cell population proceeding through different steps of the apoptotic pathway. Only cells undergoing important morphological changes in the adhesion surface are annexin-tagged. In contrast, cells progressing through apoptosis but maintaining a "flat" aspect are annexin V-negative, although it is not possible to forecast if these cells will become positive later on. These results indicate that in NIH3T3 cells PS exposure is prevented if cell shape is substantially preserved.
However, cytoskeletal changes and PS exposure are independent although strictly coupled (
In summary, our results show that identification of apoptosis in adherent cells by annexin V-FITC can introduce important biases whose entity and degree depend on the cell type under examination and on the time elapsed after stimulus application. In contrast, calcein allows checking of almost all of the pertinent morphological indexes (i.e., cell shrinkage, chromatin condensation, nuclear fragmentation, blebbing, preservation of membrane integrity) and therefore is not affected by the asynchronous progression of the population throughout apoptosis.
![]() |
Acknowledgments |
---|
Supported by CNR target project "Biotechnology."
The confocal apparatus is a facility of the Centro Interfacoltà Misure of the University of Parma.
Received for publication December 29, 1997; accepted April 8, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bevers EM, Comfurius P, Zwaal RFA (1996) Regulatory mechanisms in maintenance and modulation of transmembrane lipid asymmetry: pathophysiological implications. Lupus 5:480-487[Medline]
Bussolati O, Belletti S, Uggeri J, Gatti R, Orlandini G, Dall'Asta V, Gazzola GC (1995) Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells. Exp Cell Res 220:283-291[Medline]
Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE (1997) Geometric control of cell life and death. Science 276:1425-1428
Dall'Asta V, Gatti R, Orlandini G, Rossi PA, Rotoli BM, Sala R, Bussolati O, Gazzola GC (1997) Membrane potential changes visualized in complete growth media through confocal laser scanning microscopy of bis-oxonol-loaded cells. Exp Cell Res 231:260-268[Medline]
Frey T (1997) Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 28:253-263[Medline]
Martin SJ, Reutelingsperger CPM, McGahon AJ, Rader JA, van Schie RCAA, LaFace DM, Green DR (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182:1545-1556[Abstract]
Morris S (1990) Real-time multi-wavelength fluorescence imaging of living cells. Bio Techn 8:296-312
Pellicciari C, Bottone MG, Biggiogera M (1997) Detection of apoptotic cells by annexin V labeling at electron microscopy. Eur J Histochem 41:211-216[Medline]
Reutelingsperger CPM, van Heerde WL (1997) Annexin V, the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis. Cell Mol Life Sci 53:527-532[Medline]
Suzuki T, Fujikura K, Higashiyama T, Takata K (1997) DNA staining for fluorescence and laser confocal microscopy. J Histochem Cytochem 45:49-53
van Engeland M, Kuijpers HJK, Ramaekers FCS, Reutelingsperger CPM, Schutte B (1997) Plasma membrane alterations and cytoskeletal changes in apoptosis. Exp Cell Res 235:421-430[Medline]
van Engeland M, Ramaekers FCS, Scutte B, Reutelingsperger CPM (1996) A novel assay to measure loss of plasma membrane asimmetry during apoptosis of adherent cells in culture. Cytometry 24:131-139[Medline]
Weston N, Parish C (1990) New fluorescent dyes for lymphocyte migration studies. J Immunol Methods 133:87-97[Medline]
Wyllie AH, Kerr JFR, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251-306[Medline]
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326-1331[Abstract]