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Keywords:

  • apoptosis;
  • flow cytometry;
  • Annexin-V;
  • ISEL;
  • CD34

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background

To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death.

Methods

Apoptosis was induced in Jurkat cells by γ-irradiation, incubation with camptothecin (CPT), or cytosine β-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI− CD34+ fractions were cultured in a single-cell single-well (SCSW) assay.

Results

Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI− cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80–90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI− fractions (overall range 23–62%). Within total AnV+ and AnV+/PI− populations, only a minority of CD34+ cells showed ISEL positivity (range 4–8% and 0.8–6%, respectively). Different fractions of AnV+/PI− CD34+ cells did have clonogenic capacity.

Conclusions

PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI− CD34+ cells in the SCSW assay. Cytometry 47:24–31, 2002. © 2001 Wiley-Liss, Inc.

Each electrophoretic (EP), immunohistochemical (IH), and flow cytometric (FCM) technique detects only one specific feature of the process of apoptosis. Gel- and pulse-field EP methods detect small oligonucleosomal DNA strand breaks (multiples of 180–200 bp; DNA laddering) and large DNA strand breaks (more than 30–50 kbp), respectively, which are supposed to be relatively late and early features of programmed cell death (PCD). Unfortunately, EP techniques are not applicable for quantification of PCD. In contrast, IH techniques like in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) of DNA and FCM methods like Annexin-V (AnV)-fluorescein isothiocyanate (FITC), TUNEL, and propidium iodide (PI) can detect apoptosis of single cells.

AnV detects phosphatidylserine (PS) transposition on the outer plasma membrane, which occurs at a rather early stage of PCD during the so-called execution phase (1–6). This loss of membrane asymmetry develops downstream of the Bcl-2 checkpoint, after the disruption of the mitochondrial transmembrane potential, the release of apoptosis-inducing factor, and the activation of caspases (1). PS translocation precedes nuclear condensation, loss of membrane integrity (causing PI uptake), and cell shrinkage (6, 7). Whether PS transposition occurs before or after “the point-of-no-return” of PCD is still a matter of debate. ISEL is an IH technique described originally by Wijsman et al. (8) and modified successfully for plastic- embedded BM tissue by Mundle et al. (9). A mix of four nucleotides with DNA-Polymerase-I is used to detect specific 3′-OH ends of single-strand DNA breaks, which are found after endonuclease activation. Therefore, ISEL is detected in the phase of PCD beyond the point-of-no-return. We modified this ISEL technique and made it applicable for FCM (10, 11). We used it in combination with AnV/PI to study the kinetics of different features of PCD. Furthermore, we questioned if PS translocation under every circumstance means inevitable apoptosis by culturing sorted AnV+/PI− CD34+ cells in a single-cell single-well assay.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Materials

The T-cell leukaemia Jurkat cell line was cultured in RPMI (Gibco, Paisley, UK) with 10% fetal calf serum (FCS; Gibco) and treated in different ways to induce PCD in vitro. These cells (0.5 × 106) were either incubated with 4 ml fresh medium containing camptothecin (CPT; 2 μg/ml, Sigma, Zwijndrecht, The Netherlands) or cytosine β-D-arabinofuranoside (Ara-C; 10−4 M, Sigma), or they were γ-irradiated with 12.5 Gray (Gy) and subsequently cultured for 7 days. PCD was measured (in duplicate) by AnV/PI in time and different fractions were sorted and subsequently fixed overnight in freshly prepared 4% paraformaldehyde at 4°C. These cells were stored in 70% ethanol at –20°C for ISEL analysis at a later time.

The BM of three normal donors (D1–D3) was processed by Ficoll density separation (1.077 g/ml, Sigma) to obtain a mononuclear cell fraction. CD34+ progenitors were isolated from this fraction with directly conjugated CD34 antibody-coupled immunomagnetic beads (Dynal, Oslo, Norway). After a wash with glucose-phosphate buffered saline (G-PBS) with bovine serum albumin (BSA), these cells were resuspended in Iscove's medium, supplemented with 10% v/v heat-inactivated FCS and 10% v/v dimethylsulfoxide (DMSO) at a concentration of 0.2–0.4 × 106 cells/ml. Subsequently, the cells were cryopreserved in a temperature-controlled freezer (Kryo 10, Planerbiomed, Sunbury, Middlesex, United Kingdom) and stored in liquid nitrogen at –198°C. Cells were thawed rapidly in a waterbath of 37°C and diluted in FCS, containing 0.2 mg/ml DNAse, 4 M MgSO4, and 15 U/ml heparin. PCD analysis by FCM (AnV/PI and ISEL, in duplicate) was performed immediately after CD34 selection, after thawing, and after 4.5 h in Iscove's medium with FCS in a 5% CO2 atmosphere at 37°C.

PCD Analysis by FCM on an Epics Elite ESP (Beckman Coulter, Hialeah, FL)

For the AnV/PI procedure described in detail in Vermes et al. (3), we always used freshly obtained cells without preceding fixation. After centrifugation, the cells were washed with RPMI medium with 5% FCS. Incubation was performed with AnV-FITC (end-concentration [EC] 1.2 μg/ml, Bender Medsystems/Cordia, Leiden, The Netherlands) with an excess of calcium (2.5 mM) and PI (EC 1.6 μg/ml, Sigma) for 10 min. Jurkat cells in medium without cytotoxic treatment served as a negative control. Different AnV/PI fractions were sorted for ISEL by FCM (see Fig. 2 for the various compartments): AnV−/PI−, AnV+/PI−, AnV+/PI++, AnV+/PI+.

The modified ISEL technique for FCM (FCM-ISEL) on fixed cells was applied as follows. After washing the cells with PBS-BSA, incubation with SSC (NaCl 0.3 M, sodium citrate 30 mM, pH 7.0) was performed for 20 min at 78°C, followed by another wash in PBS-BSA. After washing with buffer (Tris HCl 50 mM, MgCl2 5 mM, β-mercaptoethanol 10 mM, BSA 0.005%, pH 7.5), incubation with DNA-Polymerase-I (20 U/ml, Promega, Madison, WI) together with 11-bio-dUTP (0.5 μM, Sigma), dATP, dCTP, and dGTP (10 μM each, Promega) was carried out at 19°C for 30 min. A wash with PBS-BSA and incubation with streptavidin-Cy5 (1.0 μg per sample) at 4°C for 30 min was performed. Finally, the cells were washed with PBS-BSA followed by FCM-ISEL. Incubation of cells without DNA-Polymerase-I served as a negative control. All AnV/PI and ISEL measurements were performed (in duplicate) on CD34+ cells within the life-gate and on Jurkat cells after setting the discriminator to exclude debris by forward and right angle scatter.

SCSW Assay

Human BM CD34+ cells were stained with AnV-FITC. An autoclone unit sorted single PI−CD34+ cells within the life-gate which were bright (AnV++: AnV fluorescence intensity ≥3), dull (AnV+: AnV fluorescence intensity 0.5–3), or negative (AnV−: AnV fluorescence intensity <0.5) for AnV in 96-well round-bottom plates (Costar, Corning, NY). Every well was checked for the presence of one single cell by inverted microscopy before culturing. Each well contained 75 μl Iscove's medium (Gibco) supplemented with 2 mM L-glutamine (Flow Laboratories, Zwanenburg, The Netherlands), streptomycin 50 mg/ml, penicillin 50 IU/ml (Gibco), 20% v/v FCS, and recombinant growth factors. This medium was supplemented with granulocyte-colony stimulating factor (20 ng/ml, Amgen, Thousand Oaks, CA), human stem cell factor (25 ng/ml, Amgen), interleukin-3 (50 ng/ml, Sandoz BV, Uden, The Netherlands), and granulocyte macrophage-colony stimulating factor (20 ng/ml, Sandoz). The plates were incubated in a fully humidified, 5% CO2 incubator at 37°C. The proliferative capacity of these CD34+ cells was assessed by counting the cells in every well at day 14 by an inverted microscope. Proliferative capacity was assessed by enumerating wells with 2 or more cells, more than 50, and more than 500 cells per 96 wells (Table 1).

Table 1. Apoptosis Measurements (mean-SD) of Life-Gated CD34+ Cells After CD34 Selection With Immunomagnetic Beads (D1) and After Viable Freeze-Thawing of CD34+ Cells (D1-3), Followed by Incubation in Medium (D1) or Plating in the SCSW Assay (D2-D3) With Size and Number (#) of Cell Aggregates per 96 Wells
D1/CD34+Life-gatedAnV−/PI−AnV+/PI−AnV+/PI+Total AnV+ISEL+
After CD34+ selection (%)37.8–2.161.9–2.10.35–0.0762.2–2.15.9
After thawing CD34+ (%)60.5–2.539.4–2.40.15–0.0739.6–2.54.6–0.5
After incubation medium (%)68.831.10.131.27.6–0.1
D2/CD34+Total (%)SCSW>2 cells>50 cells>500 cellsISEL+ (%)
AnV−/PI−75.1# aggregates181080
AnV+/PI−23.6AnV+16740.8
AnV++14114.1
AnV+/PI+1.3000
D3/CD34+Total (%)SCSW>2 cells>50 cells>500 cellsISEL+ (%)
AnV−/PI−63.0# aggregates271180
AnV+/PI−35.0AnV+14543.8
AnV++8315.4
AnV+/PI+1.5000

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The modified ISEL technique works well. FCM-ISEL without DNA-Polymerase-I served as a negative control (Figs. 1A,B). After adding DNA-Polymerase-I to CPT-treated Jurkat cells, an excellent separation between ISEL+ and ISEL− cells (Figs. 1C,D) was found.

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Figure 1. Jurkat cells treated with CPT (2 μg/ml) for 50 h and stained with the FCM-ISEL method. C,D: ISEL+ cells are positioned to the right. A,B: FCM-ISEL without DNA-Polymerase-I (serves as negative control).

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Kinetic Analysis of CPT-Induced Apoptosis in Jurkat Cells by AnV/PI by FCM

The process of PCD in time during CPT incubation (0–4–22–48–72–169 h) determined by AnV/PI is depicted in Figure 2. Fresh AnV−/PI− cells became AnV+/PI− within 4 h (mean AnV and PI fluorescence intensities of 27.3 and 0.42, respectively), after which they developed strong PI positivity (AnV+/PI++ within 22 h: mean AnV and PI fluorescence intensities of 39.1 and 63.1, respectively). All cells eventually turned into end-stage apoptosis as an AnV+/PI+ population (within 22 h and more: mean AnV and PI fluorescence intensities of 37.8 and 6.0, respectively). Figure 3 shows the time course quantity of these AnV/PI fractions. The same traverse pattern as depicted in Figure 2 was observed in Figure 3.

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Figure 2. The process of apoptosis in time of Jurkat cells during various time periods (T in hours) of CPT incubation (2 μg/ml). Fresh AnV−/PI− cells become AnV+/PI− within 4 h and develop strong PI positivity (AnV+/PI++) within 24 h. Subsequently, all these cells turn into an AnV+/PI+ population as the AnV−/PI−, AnV+/PI−, and AnV+/PI++ populations disappear.

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Figure 3. Quantification of apoptosis by AnV/PI FCM of Jurkat cells treated with CPT during various incubation periods. ▪, % AnV+/PI−; ▴, % AnV+/PI++; •, % AnV+/PI+.

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Development in Time of AnV/PI and ISEL in CPT-Treated Jurkat Cells

ISEL was performed on the AnV/PI fractions on two time points (Fig. 4). AnV− cells showed no ISEL positivity, whereas ISEL positivity rapidly increased in the AnV+/PI− fraction to almost 70–80%. ISEL positivity ultimately gained 10–20% when the AnV+/PI++ fraction was formed, after which it decreased by almost the same magnitude by turning into the end-stage AnV+/PI+ fraction.

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Figure 4. Determination of ISEL positivity (mean and SD) of the different AnV/PI fractions of Jurkat cells during 24 and 47 h of CPT incubation. Solid bars, T = 24 h; shaded bars, T = 47 h.

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If we looked more carefully at the early phase of the PCD process (0–4 h, Fig. 5) of CPT-treated Jurkat cells, an evident increment of total AnV positivity was noted between 2 and 3 h. This increment of AnV+/PI− cells was rapidly followed by a clear gain of ISEL+ cells (between 3 and 4 h) with no concomitant rise in PI.

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Figure 5. Jurkat suspension culture treated with CPT for 4 h; apoptosis measurements in duplicate depicted as mean % positive cells (and SD). Black bars, AnV+ tot; darkly shaded bars, AnV+/PI−; lightly shaded bars, ISEL+; open bars, PI+.

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PCD Development of Jurkat Cells Treated With Three Inducers of Apoptosis

As can be seen in Figure 6, the increment in time of the percentage AnV+ CPT-treated Jurkat cells was followed by a same profile of increment of the percentage ISEL+ and PI+ cells, although the differences were less pronounced as time proceeded. Quite different patterns of PCD of Jurkat cells were found during Ara-C incubation and after γ-irradiation (Fig. 6); they both showed less PCD and a more gradual increment of apoptosis. But, in general, we always observed the same pattern of phases of PCD in time (AnV+ earlier and higher than ISEL+). The difference between both apoptotic populations was not high (maximum 15–20%). Furthermore, the PI+ fraction remained in the same range as the ISEL+ fraction.

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Figure 6. The kinetic pattern of apoptosis of Jurkat cells treated with CPT, Ara-C, and γ-irradiation is quantified by three FCM techniques: AnV-FITC, ISEL, and PI. Black bars, AnV; shaded bars, FCM-ISEL; open bars, PI.

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PCD Measurements by FCM of Sorted BM CD34+ Cells: Influence of Cell Handling

In Table 1, the results of D1 of different PCD characteristics (mean %-SD) are depicted as percentages of the whole CD34+ population within the life-gate after CD34 selection, after viable freezing and thawing, and after 4 h of incubation in medium after thawing. A variable amount of cell loss is anticipated and was found after each cell handling procedure, as was proven by a gradual increment of the amount of cell debris after each procedure (5.5–12.1–13.4%, respectively). On the other hand, the percentage of CD34+ cells within the life-gate only showed a slight decrease during these procedures (70.5–70.0–67%, respectively), representing some decrease in absolute numbers of vital CD34+ cells. An unexpected high amount of total AnV positivity was found (62.2 ± 2.1%) directly after CD34 selection with antibody-coupled beads, of which only a small part showed real features of PCD (ISEL+ of 5.9% versus AnV+/PI+ of 0.35 ± 0.07%). After viable freezing and thawing, the amount of AnV+ CD34+ cells was considerably less but still remained high (39.6 ± 2.5%), whereas only a slightly lower mean ISEL+ and AnV+/PI+ level was found (4.6 ± 0.5% and 0.15 ± 0.07%, respectively). Incubation of the thawed cells in medium for 4.5 h caused some decrease in total AnV+ (±8%), which was totally attributed by a shift from the AnV+/PI− fraction to the AnV−/PI− population. The amount of AnV+/PI+ hardly changed (0.1%), whereas the amount of ISEL positivity almost doubled (7.6 ± 0.1%). Under all these circumstances, the difference between AnV+/PI− and ISEL+ populations of CD34+ cells remained at least 24% with a maximum of 55%.

Culturing Different AnV/PI Fractions of Thawed CD34+ Cells in an SCSW Assay

After thawing the sorted CD34+ cells of D2 and D3, the AnV fractions within the PI− compartment (AnV−, AnV+, AnV++) were used for SCSW assay to detect their proliferation capacity as a golden standard of viability. These AnV fractions were also sorted from the life-gate after excluding cell debris. The difference between the AnV+/PI− populations of both donors was 10–15% (Table 1), which was not correlated with their percentages of ISEL+ found within the dull and bright AnV+/PI− CD34+ cells. As AnV−CD34+ cells showed normal growth in the SCSW assay, this fraction grew approximately twice as good as their AnV+/PI− counterparts. Within this last group, the dull AnV+ cells grew considerably better than the bright AnV++CD34+ cells. AnV+/PI+ CD34+ cells showed no growth at all, as was expected.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In this study, we used Jurkat cells to observe the kinetics of three features of PCD by FCM. We also used CD34+ cells to measure apoptosis characteristics after cell handling and to investigate if AnV positivity after cell handling implied inevitable PCD. PS transposition of cells has been recognized as an evolutionary well-saved and ubiquitous marker of early PCD that is needed for cell engulfment by macrophages before their plasma integrity becomes compromised (5, 12). However, PS translocation is not unique for apoptosis because it was also observed during (secondary) necrosis (13). Therefore, we used the AnV-FITC assay with PI by time-lapse examination to differentiate among viable (AnV−/PI−), early apoptotic (AnV+/PI−), late apoptotic, and secondary necrotic (AnV+/PI++ or AnV+/PI+) cells (3, 4, 6, 7, 13–15). Furthermore, as we needed a method to distinguish between early and late PCD, we developed a modified ISEL technique for FCM (10, 11). ISEL is a variant of the TUNEL technique, but TUNEL is used much more frequently in research (16, 17). DNA-Polymerase-I used in ISEL was tested and it was able to detect DNA fragments generated by endonuclease activity in Jurkat and human BM CD34+ cells during PCD (18–21). To study apoptosis in time, we preferred the ISEL technique because it has been shown to be more specific for PCD than for necrosis compared with TUNEL (20, 22, 23). Furthermore, the TUNEL assay is prone to false positive and negative staining (15, 24). ISEL and PI detect specific DNA fragments and loss of cell membrane integrity, respectively, which are features of inevitable PCD as they occur after each other during the execution phase (1, 2, 16, 25–27). From our background of research with stem cells, we questioned if detection of AnV (within CD34+ cells) means that the point-of-no-return of PCD has been passed. This is still a matter of debate, although some evidence in favor of this hypothesis has been gathered (6, 28, 29).

In line with the above-mentioned considerations, we proved in all Jurkat experiments that membrane PS asymmetry precedes DNA fragmentation and/or membrane perturbation (Figs. 2, 3, and 5), as DNA fragmentation precedes cell membrane leakage (Figs. 4 and 5). Others (1, 4, 6, 13, 16, 17, 30–32) have obtained similar results with various techniques (AnV/PI, AnV/PI with TUNEL, AnV/TUNEL, different DNA binding dyes, morphology, comet assay, and laser scanning cytometry), but not in one setup of serial experiments in time combining three PCD-analyzing techniques with three apoptosis-inducing methods. In the experiment exposing Jurkat cells to CPT, PS expression increased after 2–3 h (AnV+/PI−) and was followed by ISEL increment by only a 1-h difference (Fig. 5). Others also found this relation in time with different techniques (28, 32–35) and it emphasizes the discrete line in time to pass the point-of-no-return regarding PCD (of this cell line under these circumstances). On the other hand, Bacsó et al. (32) proved within a CD95-induced Jurkat apoptosis model that virtually all the AnV+/PI− cells, which increased after 2 h, had apoptotic comets or remnants (as it also detects early 50-kb DNA fragments).

After carefully analyzing the process of PCD in time, an interesting pattern was observed. AnV+/PI−/ISEL− viable cells developed into AnV+/PI−/ISEL+ and subsequently AnV+/PI++/ISEL++ cells, representing a more progressive phase of apoptosis with the highest ISEL positivity. These cells turned into AnV+/PI+/ISEL+ cells, which are end-stage apoptotic and/or secondary necrotic cells, presumably characterized by more DNA disintegration, nuclear condensation, and leakage of cell and nuclear membranes causing less PI- and ISEL-positive staining (15). These results are comparable with the Nicoletti assay (3, 36) and with the report of MacNamara et al. (37) who studied HL-60 cells under similar conditions. They determined the PCD of these cells as a sub-G0/G1 peak on DNA histograms with forward and sideward scatter features reflecting cell shrinkage and the presence of apoptotic bodies.

The different PCD patterns of Jurkat cells after γ-irradiation, CPT, or Ara-C were explained by the detrimental action upon different cellular targets and by a different dose response. One should realize that the death of these cells is necrotic at high levels of insult, whereas PCD is induced at lower levels. For example, γ-irradiation promptly caused single and double DNA strand breaks, some of which were sublethal and could be repaired, some of which were lethal (causing PCD), and some of which were devastating to the cell (causing necrosis). This explains the initial combination of primary necrosis and apoptosis (of especially cells in S-phase; 38), in which the higher amount of necrosis with more PI positivity (higher than ISEL) gradually declined in order to make place for more apoptotic involvement (ISEL > PI). CPT, a DNA topoisomerase I blocker, induces DNA strand breaks of all cells (although DNA-replicating S-phase cells are more sensitive), whereas Ara-C, a pyrimidine antagonist, predominantly effects S-phase cells. Perhaps this could explain a slower increment in PCD by Ara-C compared with CPT (29, 33–35).

High numbers of AnV+/PI− of CD34+ cells within the life-gate occurred after CD34+ selection with immunomagnetic beads (D1: ±62%) and they also showed a high variability after rapidly freezing and thawing (mean AnV+/PI− of D1-D3: 33 ± 8%). It is a well-known and accepted phenomenon that a highly variable amount of stem cells is lost after CD34 selection and viable freezing and thawing. Cryopreservation in liquid nitrogen of mononucleated BM cells in 10% DMSO leads to absent trypan blue exclusion in approximately 10–15% cells and to a 25 ± 10% loss of stem cells and colony forming unit-granulocyte macrophages (CFU-GM; 39). What are the characteristics of the CD34+ cells within the life-gate regarding cell function versus apoptosis after these cell handling procedures? Cryopreservation of hematopoietic stem cells leads to high and variable AnV positivity (range 5–70%), but whether these cells are destined to die remains to be proven and should not be presumed (40). Membrane alteration or damage could be triggered by the handling during the antibody-coupled immunomagnetic bead selection or could be induced by controlled freezing (DMSO should prevent crystal formation) and rapidly thawing (DMSO can cause osmotic shock) of these cells (41). The big difference in percentage between total AnV+ and ISEL+ CD34+ cells (between 20–55%) in combination with an unchangeable low percentage in ISEL+ cells (±5%) after CD34 selection and after thawing argues strongly for a temporary membrane alteration and not for PCD. In our experiment, at least some of these AnV+ cells repaired their membrane activation-alteration and/or damage during incubation in medium (approximately 6–8% in D1), whereas other cells followed their path of PCD as ISEL was increasing. Furthermore, at least 8% (8 of 96 wells) to 15% (14 of 96) of these thawed AnV+/PI− CD34+ cells (AnV++ fraction) had proliferative capacity. On the other hand, these cells represented at least 30% (8 of 27 wells) to 78% (14 of 18 wells) of the normal plating efficiency found within these two controls in this SCSW assay. Normal plating efficiency (using AnV- CD34+ cells) is defined as the number of wells showing proliferative capacity. In contrast, the CFU-GM capacity (to form colonies of more than 40 cells as being granulocytes and macrophages) was strongly and inversely correlated with the AnV intensity of these PI− CD34+ cells. Therefore, AnV should not be used as a marker or as the only marker of apoptosis in experiments with stem cells in which physical membrane activation or alteration may be expected. PCD should be proved by distinct morphological features or by FCM techniques that detect DNA fragmentation products (15, 32). From these experiments, we conclude that in some experimental settings membrane activation and/or a low apoptotic insult was involved in causing PS exposition in a commitment phase to apoptosis. This phase of “preapoptosis” has been shown to be caspase independent and reversible if the strength of the stimulus is low and of short duration (26). A substantial fraction of AnV+/PI− BM CD34+ cells after different cell handling procedures has certainly not passed the point-of-no-return in the process of PCD, as the cells retained proliferative capacity, although to a lower extent. In analogy, cryopreservation and thawing of human spermatozoa (42) were associated with the induction of membrane PS translocation and high post-thaw levels of AnV were found even in the fractions with high sperm motility.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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