Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells

OBJECTIVE: To visualize and localize fragmented DNA strands within apoptotic cells by means of fluorescence using TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: For this experiment, lymphoid reverted cells were used as a model. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanate (TRITC). The DNA from cell nuclei was counterstained using chromomycin A3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the ability to detect DNA breaks in individual cells using TUNEL techniques and its amplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of images of TUNEL preparations and on four-dimensional (4-D) sequences of images of TUNEL-CARD preparations. RESULTS: Distribution and amplitude of fluorescent structures were characterized on dynamic sequences of images. Characterization was improved when FAMIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum of TRITC and CA3. CONCLUSION: It is possible to discriminate targets from CA3. FAMIS and TUNEL methods can be used to visualize and localize multiple DNA breaks in lymphoid reverted cells in improved methods of experimentation.     

Analysis of the fluorescence of monodansylcadaverine-positive cytoplasmic structures during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells. FAMIS was used to transform the image sequences in factor curves and images. RESULTS: By UV-CLSM, 7-keto fluorescence was detected together with MDC, which revealed morphologic cytoplasmic changes in cells. The factor images obtained from confocal image sequences emphasized the view of these results. These data are in agreement with biochemical characterizations of MDC-positive structures. CONCLUSION: The combined use of confocal microscopy and FAMIS allowed us to detect MDC-positive cytoplasmic structures in 7-keto-treated cells and to colocalize MDC and 7-keto distribution. This new method confirms the usefulness of MDC as a marker of oxysterol-induced cell death.     

Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide

OBJECTIVE: To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles). CONCLUSION: FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.     

Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.     

Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342

OBJECTIVE: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their spectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.     

Histochemical demonstration of apoptotic cells in the chicken embryo using annexin V

This study describes the use of biotinylated annexin V for the histochemical detection of apoptotic cells in cultured chicken embryos during gastrulation. This method is based on the Ca2+-dependent binding of annexin V to phosphatidylserine, a negatively charged phospholipid, located at the inner leaflet of the cell membrane in living cells. However, in the early stages of apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane and can then be recognized by annexin V. Applying this method in cultured chicken embryos during gastrulation, we obtained labelling of apoptotic cells in the three germ layers. In the epiblast and mesoblast, labelling was predominantly present in the region lateral to the primitive streak. At the level of the germinal crescent, labelled cells were also found in the epiblast. Labelled cells in the deep layer, which is a heterogeneous tissue layer composed of endophyll, sickle endoblast and definitive endobl ast, were rather scarce. The distribution of cells, as observed in this study after labelling with annexin V in light microscopy and confocal laser scanning microscopy, is consistent with distributions reported by other authors using other approaches and with our previous observations made with the TUNEL technique and by electron microscopy after fixation in a tannic acid-based fixative. The main advantages of this method over other more sophisticated methods is its easiness and rapidity of execution and the fact that both early and late stages of apoptosis are detected. © 1998 Chapman & Hall     

Surfactant protein A (SP-A) binds to phosphatidylserine and competes with annexin V binding on late apoptotic cells

The role of surfactant protein A (SP-A) in the recognition and clearance of apoptotic cells is well established, but to date, it is still not clear which surface molecules of apoptotic cells are involved in the process. Here we present evidence that phosphatidylserine (PS) is a relevant binding molecule for human SP-A. The binding is Ca²⁺-dependent and is not inhibited by mannose, suggesting that the sugar-binding site of the carbohydrate recognition domain (CRD) of SP-A is not involved. Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells, and this was consistent for Jurkat cells and neutrophils. Supporting these data, confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells. However, we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface, as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.     

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy

OBJECTIVE: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM). STUDY DESIGN: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei. RESULTS: FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained. CONCLUSION: QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.     

Detection of apoptosis using the C2A domain of synaptotagmin I

Binding of annexin V or the C2A domain of synaptotagmin I to phosphatidylserine expressed on the surface of apoptotic cells can, when labeled with appropriate probe molecules, be used to detect the presence of apoptosis using radionuclide, magnetic resonance, and optical imaging techniques. The preparation of a biotinylated C2A-GST fusion protein is described, and its capability, when used in conjunction with fluorescein-labeled streptavidin, of detecting apoptotic cells by flow cytometry is compared directly with the performance of a commercial preparation of fluorescein-labeled annexin V. Biotinylated C2A-GST, when used in conjunction with streptavidin-conjugated superparamagnetic iron oxide nanoparticles or Gd-chelate-avidin conjugates, was shown to be capable of detecting apoptotic cells using T(2)-weighted or T(1)-weighted magnetic resonance imaging experiments, respectively.     

Improved confocal characterization of targets in nuclei of cytogenetic preparations

OBJECTIVE: To show that cellular preparations requiring depth analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization and primed in situ) can be improved by regularized factor analysis of medical image sequences (FAMIS) to isolate fluorescent probes by means of intensity depth profiles of fluorochromes, to track relevant DNA sequences (cosmids and centromeres) in cell nuclei during interphase and to improve the use of cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets. STUDY DESIGN: 3D sequences of images obtained by depth displacement in a confocal microscope were first analyzed by the FAMIS algorithm, which provides factor curves. Factor images then resulted from regularization methods that improve signal/noise ratio while preserving target contours. RESULTS: Factor curves and regularized factor images helped analyze targets inside nuclei. CONCLUSION: It is possible to process preparations containing numerous spots (even when they are on different planes) to differentiate stained targets, to investigate depth differences and to improve visualization and detection.     

Bovine Lactadherin as a Calcium-independent Imaging Agent of Phosphatidylserine Expressed on the Surface of Apoptotic HeLa Cells

Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical cancer cell line HeLa has been employed as a model system to compare the topographic distribution of PS with the two PS binding proteins as adherent cells enter the apoptotic program. HeLa cells were cultured on glass-bottom Petri dishes, and apoptosis was induced by staurosporine. Fluorescence-labeled lactadherin and/or annexin V were used to detect PS exposure by confocal microscopy. Both lactadherin and annexin V staining revealed PS localized to plasma membrane rim and blebs. In addition, lactadherin identified PS exposure on long filopodia-like extensions, whereas annexin V internalized in granule-like structures. All in all, the data further delineate the differences in PS binding patterns of lactadherin and annexin V. (J Histochem Cytochem 57:907–914, 2009)     

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca²⁺, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[³⁵S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.     

Plasma Membrane Alterations and Cytoskeletal Changes in Apoptosis

During apoptosis, one of the first membrane changes that can be detected is exposure of phosphatidylserine residues at the outer plasma membrane leaflet, while early apoptosis is also accompanied by changes in the cytoskeletal organization. In this study we investigated the relationship between these two phenomena during olomoucine- and roscovitin-induced apoptosis in human lung cancer and neuroblastoma cell lines. Loss of membrane asymmetry was detected by biotin-labeled or FITC-labeled annexin V binding to negatively charged phosphatidylserine, while cytoskeletal components were visualized by immunocytochemistry. The apoptotic, annexin V-positive, cells were analyzed by flow cytometry, confocal scanning laser microscopy, and Western blotting. We report that cytokeratin and vimentin aggregation in early apoptosis occurs simultaneously with phosphatidylserine exposure and chromatin condensation. In contrast to these intermediate filament proteins, which were disassembled and proteolytically cleaved in early apoptosis, microfilaments and microtubuli were not proteolytically degraded but were found to be present as aggregated filaments in the apoptotic bodies. We also show that loss of membrane asymmetry and cytokeratin aggregation are independent processes, sinceN-ethylmaleimide-induced phosphatidylserine exposure does not cause cytokeratin disassembly. Vice versa, phorbol 12-myristate 13-acetate-induced cytokeratin filament aggregation does not result in phosphatidylserine exposure.     

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

Multiple excitation confocal analysis of targets in nuclei of cytogenetic preparations

OBJECTIVE: To demonstrate that cellular preparations requiring color analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization) can be processed by spectral analysis of fluorescent emissions by either factor analysis of medical image sequences (FAMIS) or a META confocal configuration to isolate fluorescent probes. STUDY DESIGN: Three-dimensional sequences of images obtained by spectral analysis in a META confocal microscope (Carl Zeiss SAS, Jena, Germany) were analyzed by META processing and the FAMIS algorithm, which provides factor curves. META and factor images were then the result of image-processing methods that cover emission spectra. RESULTS: Factor curves and factor or META images can help to analyze targets inside nuclei. CONCLUSION: It is possible to process preparations containing numerous spots on different colors to differentiate stained targets and to improve visualization and detection.     

Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction. Accepted: 2 June 1999     

Cellular imaging assay for early evaluation of an apoptosis inducer

The objective of this study was to propose a feasibility of a cellular imaging assay as an alternative to the conventional cytotoxicity assay, such as MTS assay, for apoptosis monitoring. As an apoptosis monitoring parameter, affinity interaction between phosphatidylserine (PS) and annexin V was chosen. First, the specific binding affinity between annexin V and PS in phospholipid bilayers consisting of various molar (0–15%) composition of PS was measured using a surface plasmon resonance biosensor. As PS composition increased, the binding level of annexin V increased proportionally. Second, various concentrations (0.1–10 μM) of staurosporine were used as to induce apoptosis and introduced to MCF-7 breast carcinoma cells. The cellular fluorescence images from annexin V-FITC conjugate were obtained by confocal microscopy, and their fluorescence intensities were quantified by image scanning. Dose–apoptosis (or cell death) relationships were very similar to those from MTS and FACS assays. In summary, our cellular imaging method could serve as a quicker and simpler alternative to MTS (end point assay) and FACS (flow cytometry) to screen potential apoptosis inducers.     

Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy.

BACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy. METHODS: Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths. RESULTS: Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image. CONCLUSIONS: Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.     

Calcium,leukocyte cell death and the use of annexin V: fatal encounters

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane’s outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results. Uriel Trahtemberg and Mizhir Atallah contributed equally to this publication.