Microparticles from apoptotic platelets promote resident macrophage differentiation

Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PM ap). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PM ap formation. Flow cytometry and scanning electron microscopy demonstrated that PM ap display activated integrins and interact to form microparticle aggregates. PM ap were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PM ap promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin a M b 2), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PM ap polarized the cells into resident M2 monocytes. Cells treated with PM ap actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PM ap stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PM ap with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes. The presence of microparticles in platelet-rich plasma has first been described in the late 60s, where these were designated as platelet dust. 1 Since that time, the role of platelet-derived microparticles in the (patho)physiology of thrombosis and haemostasis has been well established. Circulating platelet microparticles in normal peripheral blood support low-grade thrombin generation. 2 After surgery and under thrombotic conditions, the levels of microparticles in plasma can significantly increase, 3,4 and this has been associated with the pathologies of cardiovascular diseases, 5 inflammation and atherosclerosis. The formation of these micro-particles is considered to be a consequence of platelet activation with potent agonists, which indeed give rise to massive shedding of 'activation-induced' microparticles with a strong procoagulant potential in vitro. 6 Platelets circulate in blood for 9–10 days, and then are cleared in the spleen or liver following an apoptotic-like process. 7,8 This age-induced platelet apoptosis also leads to shedding of microparticles but, markedly, this process is not accompanied by signs of platelet activation. 9,10 Accordingly, there is a continuous formation of apoptosis-induced platelet microparticles (PM ap) in …

The presence of microparticles in platelet-rich plasma has first been described in the late 60s, where these were designated as platelet dust. 1 Since that time, the role of platelet-derived microparticles in the (patho)physiology of thrombosis and haemostasis has been well established. Circulating platelet microparticles in normal peripheral blood support low-grade thrombin generation. 2 After surgery and under thrombotic conditions, the levels of microparticles in plasma can significantly increase, 3,4 and this has been associated with the pathologies of cardiovascular diseases, 5 inflammation and atherosclerosis. The formation of these microparticles is considered to be a consequence of platelet activation with potent agonists, which indeed give rise to massive shedding of 'activation-induced' microparticles with a strong procoagulant potential in vitro. 6 Platelets circulate in blood for 9-10 days, and then are cleared in the spleen or liver following an apoptotic-like process. 7,8 This age-induced platelet apoptosis also leads to shedding of microparticles but, markedly, this process is not accompanied by signs of platelet activation. 9,10 Accordingly, there is a continuous formation of apoptosis-induced platelet microparticles (PM ap ) in blood from ageing, seemingly resting platelets. Phagocytes, including monocytes, dendritic cells and macrophages, are responsible for the clearance of apoptotic platelets and also PM ap from the circulation. 11 Conditions stimulating platelet apoptosis or suppression of phagocytosis will hence increase the plasma levels of PM ap , which may aggravate inflammation-linked disorders like atherosclerosis.
Much attention has been paid to the microparticles that are generated from activated platelets. For instance, their high procoagulant potential has been characterized, 12 their proteome with many bioactive compounds -including CCL5, CXCL4 and CXCL7 -has been unraveled 13,14 and they have recently been shown to enhance the vasoregenerative potential of angiogenic early outgrowth cells. 15 Instead, there is very little knowledge of biological effects of the 'spontaneously' formed apoptosis-induced microparticles, the PM ap .
Here, we hypothesized that PM ap have an immunomodulating role by interacting with leukocytes, in particular monocytes. We aimed to characterize this role by determining short-and long-term effects of the interactions of PM ap with monocytic cells and primary monocytes.

Results
Interactions of monocytic cells with PM ap . Microparticlecontaining platelet-free plasma was isolated from stored platelet-plasma concentrates (5 days of storage) in which shed apoptotic microparticles accumulate time-dependently without platelet activation 10 (Figure 1a, left panel), as visualized by flow cytometry using CD61 (glycoprotein (GP)IIIa) expression as a platelet-specific marker (Figure 1a, right panel). The plasma was microscopically checked for the presence of PM ap and absence of intact platelets. Using a combination of micropore filtering (0.8 mm) and ultracentrifugation to remove intact platelets, PM ap were isolated and characterized by flow cytometry, with freshly isolated platelets as a reference. The PM ap fraction mostly contained particles of o1 mm in size, but also appreciable numbers of larger size particles (Figure 1b). These larger particles may be aggregated PM ap , as scanning electron microscopy revealed the presence of single as well as aggregated microparticles (Figure 1c). Flow cytometry characterization further indicated that CD61 expression was lower in PM ap than in resting platelets (compatible with the size difference), and that the activated conformation of GPIIb/IIIa (detected with PAC1 monoclonal antibody (mAb)) was higher in PM ap (Figures 1d and e). In PM ap the expression of markers of strong activation, for example, CD62P (P-selectin) and phosphatidylserine (annexin A5 binding), were increased in comparison to resting platelets. Together, this indicated that PM ap assume an activation state with procoagulant phospholipids and activated integrins, thus explaining their ability to form aggregates.
We then examined the ability of PM ap to bind to THP-1 cells, an established monocytic cell line. Electron microscopy

Figure 1
Characterization of apoptosis-induced platelet microparticles. (a) Spontaneous generation of PM ap in platelets stored in RPMI þ 5% FBS at 37 1C for 1-7 days (left panel) and flow cytometry visualization of platelets and PM ap (right panel, marked areas, AG: aggregates) and (b) of PM ap isolated from 5-day-old platelet concentrates. Staining was with FITC-labeled anti-CD61mAb, and 1 mm and 6 mm beads were added as size markers (in frames). (c) Electron microscopy images of platelets and of PM ap , forming small aggregates (arrows and right panel). (d) Flow cytometry detection for resting platelets (plts) and PM ap of expression levels of GPIIb/IIIa (FITC anti-CD61mAb), activated GPIIb/IIIa (FITC PAC1mAb), P-selectin (FITC-anti-CD62PmAb) and phosphatidylserine (APC-annexin A5). Mean ± S.E.M. (n ¼ 4-6). *Po0.05 versus platelets. (e) Representative histograms of the flow cytometry analysis, filled histogram: (isotype) control, dashed line: platelets, solid line: PM ap detected multiple PM ap on the surface of THP-1 cells coincubated with the microparticles (Figure 2a). Flow cytometry using anti-CD61mAb indicated that the PM ap binding to cells increased between 30 min and 70 min (Figures 2b and c). At later time points, CD61 binding sites on the cell surface decreased again. This suggested that the PM ap were internalized by endocytosis, which could be confirmed by confocal microscopy revealing PM ap localized at the surface and in the cytoplasm of the THP-1 cells (Figure 2d and Supplemental movie). Binding was completely absent after heat treatment of PM ap (56 1C for 30 min, data not shown), indicating the necessity for active adhesion molecules on the PM ap surface for interaction with monocytic cells. To determine whether PM ap contain biological activity towards blood cells, we used a Transwell migration assay detecting chemotactic effects on THP-1 cells. Strikingly, PM ap evoked robust cell migration, which was comparable with the reference chemokine, CCL2 (Figure 2e), indicating a strong chemotactic potential of the microparticles. To further investigate the potential mediators derived from PM ap , we employed blocking antibodies against CCL5 and CXCL7, two platelet-derived chemokines that have been shown to mediate mononuclear cell (MC) recruitment. 16 The chemotaxis of THP-1 cells toward PM ap could be effectively blocked with antibodies against CCL5, whereas anti-CXCL7 or isotype antibodies showed no effect (Figure 2e). This suggests that CCL5 is a major factor that controls monocyte migration in the direction of PM ap .
PM ap -induced adhesion of monocytic cells under static and flow conditions. Experiments were conducted to determine the functional consequences of monocytic cell interactions with PM ap . Preincubation of THP-1 cells with PM ap (44 h) caused a marked increase in stable adhesion to fibronectin-coated wells under static conditions. This effect was similarly effective to that of incubation with phorbol myristate acetate (PMA; Figure 3a). It tended to increase with higher counts of PM ap , whereas supernatants from PM ap preparations were inactive. Also, preincubation with resting platelets, added at similar counts, did not affect the adhesion. As platelets spontaneously shed PM ap , the generation of PM ap during the course of the coincubation experiments with resting platelets might influence our findings (Figure 1a). In order to exclude potential effects from these spontaneously generated PM ap , we first determined the amount of PM ap formed after 7 days relative to the 10/1 ratio of PM ap that we commonly used in our experiments (Figure 3b). In a subsequent adhesion assay, we compared the functional effects of this amount of PM ap with the 10/1 ratio of PM ap . As expected, no effects of this minor amount of PM ap were observed ( Figure 3c).
The effect of PM ap on adhesion of primary monocytes was studied under physiological conditions of low-shear flow over a confluent monolayer of human endothelial cells. 17 Coincubation with PM ap (44 h) dramatically stimulated the adhesion of isolated monocytes to the endothelium, whereas coincubation with platelets at similar counts had no effect  PM ap -induced differentiation to professional resident phagocytes. Changes in monocytic cell surface receptors were studied by flow cytometry after short-term (44 h) or long-term (7 days) incubation with or without PM ap . The presence of PM ap gradually increased the expression levels of CD11b (integrin a M b 2 ), CD14 (lipopolysaccharide co-receptor) and CD31 (platelet endothelial cell adhesion molecule-1) in similar ways for THP-1 cells ( Figure 4a) and for primary monocytes (Figure 4b). The presence of PM ap downregulated the expression of CCR2 (CCL2 receptor) both on THP-1 cells and monocytes after 44 h, although the expression of this receptor recovered after 7 days. By contrast, PM ap significantly increased the level of CCR5 (CCL5 receptor) in THP-1 cells, but not in primary monocytes, and increased the level of CXCR4 (CXCL12 receptor) both in THP-1 cells and primary monocytes (Figures 4a and b). Jointly this suggested that PM ap -treated monocytic cells tend to assume a resident (M2) monocyte phenotype, which previously has been defined as CD14 þ CD16 þ CCR2 À CCR5 þ CXCR4 þþ . 18 Expression levels of CD16 (FcgRIII receptor) remained unchanged in our experiments (Figures 4a and b).
Prolonged incubation of THP-1 cells with PM ap stimulated spreading of the cells on a fibronectin surface (Figure 5a).
Spreading involved the formation of filopods and lamellipods, which appeared as actin filament bundles upon staining with the actin probe, phalloidin (Figure 5a). It was also found that the presence of PM ap essentially annulled the proliferation of THP-1 cells (Figure 5b). Control staining with ethidium bromide showed that the non-viable cells remained at 6-7% after treatment with PMA, PM ap or resting platelets ( Figure 5c). Collectively, these findings supported the conclusion that THP-1 cells in the presence of PM ap develop to a resident, non-proliferating phagocytic phenotype.
Further experiments were carried out to confirm the differentiation into professional phagocytic cells. Prolonged, 7-day incubation of THP-1 cells with PM ap , but not with resting platelets, increased the uptake of oxidized low-density lipoprotein (oxLDL; Figure 5d). In agreement with this, the surface expression of the respective oxLDL and LDL receptors CD36 and CD68 (macrophage differentiation markers) was increased in the presence of PM ap (Figure 5e). Similarly, incubation of primary monocytes with PM ap for 7 days increased the CD36 level, but this period appeared to be too short to alter the CD68 expression ( Figure 5f).
PM ap -induced secretory properties of monocytic cells. Resident phagocytic monocytes are characterized by their capability to secrete matrix-degrading proteinases and reactive oxygen species. 19 Indeed, incubation of THP-1 cells with PM ap , but not resting platelets, for 2-7 days resulted in the production of functional matrix metalloproteinase (MMP)9, as was shown by zymography (Figure 6a). Quantification of the zymograms indicated a time-dependent increase of MMP activity after PM ap incubation, which was similarly high as that of incubation with PMA after 7 days (Figure 6b). This time effect also indicated that MMP derived from PM ap and platelets 20 themselves did not measurably contribute to the zymographic activity of supernatants. Furthermore, PM ap incubation markedly enhanced the release of hydrogen peroxide (H 2 O 2 ) from both monocytic cells (Figure 6c) and primary monocytes (Figure 6d).
To screen for the secretion of cytokines and chemokines, primary monocytes were incubated for 44 h with PM ap or resting platelets, after which cell supernatants were analyzed for the presence of a panel of cytokines using a semiquantitative commercial array kit (Figure 7a). Markedly, in comparison to platelets, the presence of PM ap stimulated the release of several cytokines and other factors known to be proinflammatory, such as C5a, CCL5 and interleukin-23, and to a lesser extent of macrophage migration inhibitory factor, soluble CD40 ligand and soluble intercellular adhesion molecule 1. Furthermore, the monocytes produced several factors that participate in immunomodulating and regenerative processes rather than in inflammation, such as granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). To determine the most abundantly secreted relevant cytokines in a more quantitative manner, we performed an enzyme-linked immunosorbent assay (ELISA) for C5a, GM-CSF, interferon-g (IFN-g) and tumor necrosis factor a (TNFa). Although secreted IFN-g levels were below detection limits, the ELISA measurements largely confirmed the induction of monocyte C5a, GM-CSF and TNFa secretion by PM ap , albeit that platelets more potently induced GM-CSF production in monocytes than PM ap (Figure 7b).

Discussion
In the present paper, we have identified a potent modulating role of PM ap on monocyte functions. These microparticles, formed by ageing platelets in an apoptosis-like process, appear to display a characteristic 'pro-adhesive' membrane surface with integrins (GPIIb/IIIa) in the activated conformation and exposure of phosphatidylserine, which may facilitate the interaction with monocytes. In the literature, the formation of platelet-leukocyte coaggregates is studied extensively, for example, following activation of the platelet protease-activated receptor (thrombin receptor) and P2Y 12 receptors. Very little, however, is still known about the formation of platelet microparticle-leukocyte complexes. The present study is the first to demonstrate the role of platelet microparticles, produced from ageing, and apoptotic platelets in leukocyte function and differentiation. Jointly our results show that PM ap interacting with monocytic cells and primary monocytes evoke potent effects in achieving cell polarization and differentiation. In our implemented model of coincubation of platelets and PM ap with monocytic cells in vitro, we took effort to exclude confounding effects of PM ap generated by platelets during the long-term coculture process. Indeed, the amount of spontaneously generated PM ap by platelets was found to be functionally negligible.
The precise signaling pathways that are triggered in monocytes by PM ap still need to be identified. One conceivable mechanism supported by our findings is triggering via chemokines such as CCL5, which is carried and secreted by activated platelets 16,21,22 and activation-induced platelet microparticles. 14,23 Such a mechanism explains the chemotactic effect of PM ap causing monocytic cell migration and has recently been implied in the modulation of T-cell differentiation by platelets. 24 In addition, the PM ap -induced activation of monocytes likely relies on specific molecular interactions with receptors on the microparticle surface, including lipids (phosphatidylserine), 25,26 adhesive receptors (for example, activated GPIIb/IIIa) and GP counter-receptors (P-selectin), similar to those identified for monocyte activation by platelets. 22,27 Furthermore, also endocytosis of PM ap following adhesion may force the monocytes to enter specific differentiation pathways. Interestingly, it appeared that the PM apinduced adhesion to monocytic cells was abolished by cell treatment with wortmannin, suggesting a role of phosphoinositide 3-kinase signaling pathways in the monocyte activation (E. Vasina, unpublished data and Barry et al. 28 ).
Circulating monocytes appear to form different subsets with discrete properties and functions. A conventional division is into 'proinflammatory' M1 monocytes, which secrete proinflammatory cytokines and have a cytotoxic role, and 'resident' M2 monocytes, which participate in tissue remodeling, wound healing and angiogenesis. 29 The M2 monocytes tend to differentiate into professional phagocytes characterized by potent endocytotic activity. 18 Knowing that the formation of resident monocytes is accompanied by downregulation of CCR2 and upregulation of CCR5 and CXCR4 surface receptors, we observed a marked potential of PM ap to differentiate monocytes in the direction of M2 cells and professional phagocytes. This was confirmed by an increased expression of adhesive integrins (CD11b), of the respective LDL and oxLDL receptors, CD68 and CD36, and a higher uptake of oxLDL. In support of the notion that M2 monocytes preferentially differentiate into dendritic cells, 18  PM ap -stimulated monocytes, a stimulus for differentiation to dendritic cells. 30 The present findings likely have important implications for our understanding of the pathology of atherosclerosis. Initial support for a proatherogenic role of PM ap -activated monocytes comes from their enhanced adhesion to endothelial cells under flow conditions that precedes plaque infiltration and is particularly relevant in early stages of atherosclerosis. 16 This increased adhesion is likely mediated by receptors on the microparticles or by an increased expression of adhesive monocyte receptors (for example, CD11b, CD31) 22,31 and may be further augmented by the expression of chemokines such as CCL5. 23 In addition, the monocyte subset LyC lo CCR2 À CCR5 þ was shown to use the CCL5 receptor CCR5 when entering atherosclerotic plaques in mice, 32 and the blocking of CCR5 led to a reduction of atherosclerosis in several studies. 33 Analogously, in our experiments, the polarization of human monocytes by PM ap into the CD14 þ CD16 þ CCR2 À CCR5 þ CXCR4 þþ phenotype is suggestive for the formation of a proatherogenic monocyte subset, residing in human plaques and aggravating atherosclerosis. This suggestion is supported by the observation that PM ap -activated monocytic cells upregulate the expres-sion of phagocyte markers (CD14, CD36, CD68), and massively release MMP and H 2 O 2 , which are factors that constitute to plaque destabilization and eventual rupture, a clinically precipitating event in atherosclerotic disease.
Earlier studies have shown that the production of PM ap is a continuous process in stored platelets in the apparent absence of platelet activation. 10 In the circulation, like other apoptotic bodies, these microparticles will be actively removed by binding and phagocytosis. 11 Conceivably, this removal is mediated by interactions of PM ap with leukocytes, particularly monocytes. Indeed, coaggregates of platelet fragments and monocytes could be detected in blood from healthy subjects (E. Vasina, unpublished data). On the other hand, such interactions may contribute to long-term leukocyte differentiation, as we have pointed out in this paper, and can even be a polarizing factor in the development of resident monocytes. However, further work needs to be done to demonstrate that PM ap -primed monocytes differentiate into M2 cells and resident macrophages/dendritic cells in vivo.
The present results even suggest that PM ap have an immunomodulating potential by stimulating their own phagocytic removal, as CD11b, CD14 34  is confirmed by the plethora of proinflammatory and noninflammatory cytokines, including G-CSF, GM-CSF and TNFa, that are secreted by PM ap contact with monocytes. In conclusion, this is the first study to demonstrate that microparticles spontaneously arising from apoptotic platelets are able to promote monocytes towards a resident phagocytic phenotype, changing their behavior and activation state, thus, presenting a novel mechanism in which platelets and their products might influence the progression of atherosclerotic disease.

Materials and Methods
Monocytic cells and primary monocytes. Human acute monocytic leukemia THP-1 cells were cultured in RPMI-1640 medium with L-glutamine and 10% fetal bovine serum (FBS). MCs were obtained from buffy coats of peripheral blood from healthy human donors who gave informed consent. Monocytes were separated from neutrophils by Ficoll density gradient centrifugation and further isolated by negative selection using a MACS Monocyte isolation kit II (Miltenyi Biotech, Bergisch Gladbach, Germany), according to the manufacturer's protocol. Purity of the monocyte preparation was analyzed by flow cytometry based on cell forward scatter and side scatter and amounted to 497%.
Platelet-derived microparticles and washed platelets. PM ap were isolated from platelet concentrates in plasma, which were stored for 5 days under standard blood bank conditions (Uniklinikum Aachen, Germany). Platelets were removed by rapid centrifugation at 4000 Â g for 5 min. The platelet-free plasma preparations were centrifuged at 20 000 Â g for 60 min. Pellets containing PM ap were resuspended in Hepes buffer pH 7.45 (136 mmol/l NaCl, 10 mmol/l Hepes, 2.7 mmol/l KCl, 2 mmol/l MgCl 2 , 0.1% bovine serum albumin and 0.1% glucose), filtered through a 0.8-mm filter in order to remove residual platelets, and pelleted again at 20 000 Â g for 40 min. Supernatants were used for control experiments. After resuspension of the pellet in Hepes buffer, PM ap labeled with anti-CD61-FITC mAb (BD Biosciences, Heidelberg, Germany) were counted by flow cytometry in the presence of fixed numbers of 6 mm calibration beads (BD Biosciences). Event sizes were estimated by addition of 1 mm and 6 mm fluorescent beads (Polysciences, Eppelheim, Germany). The CD61-positive events o6 mm were counted as PM ap . Suspensions of PM ap were directly used or snap-frozen in liquid nitrogen. The isolated PM ap were virtually negative for the exosome marker CD63.
Platelets were isolated from freshly obtained human blood, as described. 35 Flow cytometric determination of cell surface markers. Using flow cytometry on a fluorescence-activated cell sorting Canto II (BD Biosciences), platelets and PM ap were characterized for surface protein levels with fluorescently labeled antibodies against CD61, CD62P or activated GPIIb/IIIa (PAC-1mAb; BD Biosciences). Expression of phosphatidylserine was probed with APC-annexin A5 (BD Biosciences). THP-1 cells or monocytes in suspension were stained (30 min, 4 1C) with mouse anti-human antibodies against CD11b, CD31 (Sigma, St. Louis, MO, USA); CD14, CD16, CCR2, CCR5, CXCR4 (BD Biosciences); or CD36 (ImmunoTools, Friesoythe, Germany) and prepared as described before. 36 Intracellular CD68 was detected in cells fixed with paraformaldehyde, permeabilized with 0.5% Triton-X-100, and stained with mouse anti-human CD68mAb (BD Biosciences). Corresponding negative control antibodies were IgG1 (Sigma); IgG2b, k (BD Biosciences) or IgG2a (ImmunoTools) and used at recommended dilutions. Expression levels are presented as geometric means of fluorescence histograms, corrected for the values obtained with isotype control antibodies and as representative histograms (FlowJo software, Tree Star, Inc., Ashland, OR, USA).
Adhesion of monocytic cells. Adhesion of THP-1 cells was measured in RPMI-1640 medium supplemented with 5% FBS, as described. 37 Cells treated with vehicle, PMA (10 ng/ml, Calbiochem, Darmstadt, Germany), platelets or PM ap , were left to adhere to fibronectin-coated wells at 37 1C. At indicated times, vital cells were labeled with BCECF acetoxymethyl ester (1 mg/ml, Sigma) for 1 h, after which fluorescence was measured of all cells and of adherent cells after a wash, using a Spectra FluoPlus reader (Tecan, Männedorf, Switzerland). Adhesion was expressed as percentage of fluorescence of all cells. Incubations were performed in triplicate. In parallel experiments, labeled cells were scraped from wells and counted via calibrated flow cytometry, with essentially the same results. Adhesion of primary monocytes to a monolayer of human aorta endothelial cells (passage 6-7) under flow conditions (0.1 ml/min, 1.5 dynes/cm 2 ) was measured by microscopic video imaging. 17 Cells were preincubated with Hepes buffer (vehicle), PM ap or platelets for 44 h at 37 1C. During perfusion adherent monocytes were counted in at least five random microscopic fields.
Monocyte cell function assays. Proliferation of THP-1 cells was measured after culturing in RPMI-1640 medium with 5% FBS at 37 1C. Cells were seeded into 96well plates (3 Â 10 4 cells/well, or 1.5 Â 10 5 cells/ml), coated with fibronectin and stimulated, as described. At indicated times, adherent cells were labeled with DAPI (0.5 mg/ml for 1 h at 37 1C), and fluorescence was measured in a Spectra FluoPlus plate reader. Calibrations with known cell counts were performed to convert fluorescence values to cell numbers. In parallel, fractions of viable cells were determined microscopically by staining viable cells with calcein acetoxymethyl ester (1 mg/ml) and counter-staining leaky, non-viable cells with ethidium bromide (1 mg/ml). Fractions of