In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease. When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. Download video file.^{(78M, mp4)}     

Xenopus neural crest cell migration in an applied electrical field

Xenopus neural crest cells migrated toward the cathode in an applied electrical field of 10 mV/mm or greater. This behavior was observed in relatively isolated cells, as well as in groups of neural crest cells; however, the velocity of directed migration usually declined when a cell made close contact with other cells. Melanocytes with a full complement of evenly distributed melanosomes did not migrate of their own accord, but could be distorted and pulled by unpigmented neural crest cells. Incompletely differentiated melanocytes and melanocytes with aggregated melanosomes displayed the same behavior as undifferentiated neural crest cells, that is, migration toward the cathode. An electrical field of 10 mV/mm corresponded to a voltage drop of less than 1 mV across the diameter of each cell; the outer epithelium of Xenopus embryos drives an endogenous transembryonic current that may produce voltage gradients of nearly this magnitude within high-resistance regions of the embryo. We, therefore, propose that electrical current produced by the skin battery present in these embryos may act as a vector to guide neural crest migration.     

Mechanism of inhibition of the proximal tubular isotonic fluid absorption by polylysine and other cationic polyamino acids.

The present study was initiated with the hope of clarifying the role of negative charges in the luminal brush border membrane in the overall process of trans-epithelial isotonic sodium and water absorption. Using micropuncture techniques, cationic polyamino acids such as polylysine (mol wt 100,000, 17,000 and 1,500-5,000, 1 mg/ml), tetralysine, polyornithine (mol wt 100,000, 1mg/ml), polyethyleneimine (2 mg/ml), polymyxin B (2 mg/ml), protamine sulfate (25 mg/ml) and histone (0.5 mg/ml) were perfused through the segments of rat kidney proximal tubule for 30 sec to 2 min. The rate of isotonic fluid absorption was measured before and after each perfusion with the Gertz's split drop method using Ringer's solution as a shrinking drop. Polylysine 100,000 and 17,000 and polyornithine were the most potent, inhibiting isotonic reabsorption by 93%. The sequence of inhibitory effect was: polylysine 100,000 congruent to polyornithine 100,000 congruent to polylysine 17,000 greater than polyethyleneimine greater than polylysine 1,500-5,000 congruent to polymyxin B greater than protamine sulfate congruent to histone. In contrast, tetralysine (2 mg/ml) showed no inhibitory effect. Electrical potential difference (p.d.) of the proximal tubular cells was destroyed within 10 sec of luminal perfusion with polylysine 100,000 (1 mg/ml). Simultaneously with the drop in p.d., electrical resistance of the luminal brush border membrane was nearly totally eliminated, whereas transepithelial input resistance remained unaltered. Furthermore, trypan blue dye was taken up by polylysine 100,000-perfused tubular cells but not by normal cells. Expanding drop analysis (mannitol solution as a split drop) was performed as a screening test to examine if the permeability for water and sodium in the lateral paracellular pathway is altered by polylysine 100,000. No significant difference was observed in the velocity of split drop expansion between untreated and polylysine-perfused tubules. A lower concentration of polylysine 100,000 (0.1 mg/ml) showed a much less inhibitory effect on fluid absorption and on cell p.d. These observations indicate that the strong inhibition on proximal tubular fluid absorption exerted by polylysine and perhaps also by other cationic polyamino acids is due not to modification of membrane negative charges but to the lysis of tubular cells by these polycations.     

An analytical model of the hydraulic aspects of stomatal dynamics

An analytical model of the hydraulic aspects of stomatal dynamics is formulated in this paper. The model consists of a coupled system of non-linear, ordinary differential equations, written in terms of water potentials, hydrostatic pressures, osmotic potentials, water vapor resistances and water fluxes. The model is validated by comparisons with the experimental literature. Numerical solutions of the model show qualitative agreement with most known stomatal responses.Stomatal opening in the model is dependent on the interaction of the guard and subsidiary cells in the following manner. Pore opening is initiated by a rise in the guard cell hydrostatic pressure. As the stomate opens, transpiration increases, causing the cell wall water potential to drop. The drop in cell wall water potential then causes the subsidiary cell pressure to drop, opening is accelerated, and the stomate literally “pops” open. Simulated opening proceeds in two distinct phases: a stress phase and a motor phase. During the stress phase, guard cell pressure rises but the pore remains closed. The motor phase commences when the guard cell pressure has risen sufficiently to initiate pore opening, beyond which point opening progresses rapidly.Hydropassive stomatal movements are found to be insufficient to regulate water loss at low leaf water potentials. Stable, hydraulically-based oscillations in stomatal aperture are shown in the model by the existence of a stable limit cycle. The period of these oscillations is strongly influenced by the cell membrane hydraulic conductivity. An increased conductivity results in a shorter period oscillation. Environmental conditions promoting oscillatory behavior are in qualitative agreement with the experimental literature.     

Colony,hanging drop,and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.     

The oscillatory behavior of pancreatic islets from mice with mitochondrial glycerol-3-phosphate dehydrogenase knockout

Glucose stimulation of pancreatic beta cells induces oscillations of the membrane potential, cytosolic Ca(2+) ([Ca(2+)](i)), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca(2+) have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca(2+)](i). In the present study, we compared different types of oscillations in beta cells from wild-type and mGPDH(-/-) mice. In clusters of 5-30 islet cells and in intact islets, 15 mM glucose induced an initial drop of [Ca(2+)](i), followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca(2+)] rise, were similar in wild-type and mGPDH(-/-) mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH(-/-) beta cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca(2+)](i) during imposed oscillations induced by pulses of high K(+) or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca(2+)](i) in beta cells similarly increased NAD(P)H fluorescence in control and mGDPH(-/-) islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca(2+)](i) and secretory responses in mGPDH(-/-) islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of beta cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca(2+).     

A long-wave absorbing form of chlorophyll a responsible for the “red drop” in fluorescence at 298 °K and the F723 band at 77 °K

When Chlorella cells are ruptured at pH 4.6 by sonication in air, its absorption spectrum can be best explained if one assumes that a long-wave chlorophyll a form (Chl a 693) is preferentially destroyed. Using these preparations, and comparing them with the algal suspension and the sonicates prepared at pH 7.8 under argon, we make the following conclusions: (a) The red drop beginning at about 675–680 nm in the action spectrum^* of fluorescence at 298 °K must be due to the presence of a non-(or weakly) fluorescent form of chlorophyll a. We suggest that this form is Chl a 693. The red drop is absent in the aerobic sonicates. (b) The red drop in fluorescence in whole algal cells is not due to any errors in absorption measurements; this drop is clearly present in the anaerobic sonicates. (c) The emission band at 723 nm, discovered by in whole Chlorella cells at 77 °K, may be due to increased fluorescence efficiency of Chl a 693 at low temperature; the F723 band is absent in aerobic sonicates.     

The effect of agitation intensity on alkali-catalyzed methanolysis of sunflower oil

The sunflower oil methanolysis was studied in a stirred reactor at different agitation speeds. The measurements of drop size, drop size distribution and the conversion degree demonstrate the effects of the agitation speed in both non-reaction (methanol/sunflower oil) and reaction (methanol/KOH/sunflower oil) systems. Drop size distributions were found to become narrower and shift to smaller sizes with increasing agitation speed as well as with the progress of the methanolysis reaction at a constant agitation speed. During the methanolysis reaction, the Sauter-mean drop diameter stays constant in the initial slow reaction region, rapidly decreases during the fast reaction period and finally reaches the equilibrium level. Due to the fact that the interfacial area increases, one can conclude that the rate of reaction occurring at the interface will also be enhanced progressively. The "autocatalytic" behavior of the methanolysis reaction is explained by this "self-enhancement" of the interfacial area, due to intensive drop breakage process.     

Numerical analysis of the deformation of an adherent drop under shear flow

The adhesion of leukocytes to substrates is an important biomedical problem and has drawn extensive research. In this study, employing both single and compound drop models, we investigate how hydrodynamics interacts with an adherent liquid drop in a shear flow. These liquid drop models have recently been used to describe the rheological behavior of leukocytes. Numerical simulation confirms that the drop becomes more elongated when either capillary number or initial contact angle increases. Our results show that there exists a thin region between the drop and the wall as the drop undergoes large stretching, which allows high pressure to build up and provides a lift force. In the literature, existing models regard the leukocyte as a rigid body to calculate the force and torque acting on the drop in order to characterize the binding between cell receptors and endothelial ligands. The present study indicates that such a rigid body model is inadequate and the force magnitude obtained from it is less than half of that obtained using the deformable drop models. Furthermore, because of its much higher viscosity, the cell nucleus introduces a hydrodynamic time scale orders of magnitude slower than the cytoplasm. Hence the single and compound drops experience different dynamics during stretching, but exhibit very comparable steady-state shapes. The present work offers a framework to facilitate the development of a comprehensive dynamic model for blood cells.     

Induction of osteoprogenitor cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal cells

Bone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and β-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3–4 and 10–11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 μg/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 μg/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day-21 mineralization and only the high dose, 10.0 μg/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 μg/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.     

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.     

Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions.

Using the relations between pellet structure and electric properties derived from the preceding paper, the responses of rabbit erythrocyte pellets to osmotic or colloidal-osmotic effects from exchanged supernatants and from electroporation were investigated. Changing the ionic strength of the supernatant, or replacing it with dextran or poly(ethylene glycol) solutions, caused changes of Rp according to the osmotic behavior of the pellet. Rp was high and ohmic before electroporation, but dropped abruptly in the first few microseconds once the transmembrane voltage exceeded the membrane breakdown potential. After the initial drop, Rp increased as a result of the reduction of intercellular space. Rp increased regardless of whether the pellets were formed before or immediately after the pulse, indicating that porated cells experienced a slow colloidal-osmotic swelling. The intercellular or intermembrane distances between cells in a pellet, as a function of osmotic, colloidal-osmotic, and centrifugal pressures used to compress rabbit erythrocyte pellets, were deduced from the Rp measurement. This offered a unique opportunity to measure the intermembrane repulsive force in a disordered system including living cells. Electrohemolysis of pelleted cells was reduced because of limited swelling by the compactness of the pellet. Electrofusion was observed when the applied voltage per pellet membrane exceeded the breakdown voltage. The fusion yield was independent of pulse length greater than 10 microseconds, because after the breakdown of membrane resistance, voltage drop across the pellet became insignificant. Replacing the supernatant with poly(ethylene glycol) or dextran solutions, or coating pellets with unporated cell layers reduced the colloidal-osmotic swelling and hemolysis, but also reduced the electrofusion yield. These manipulations can be explored to increase electroloading and electrofusion efficiencies.     

Neuroanatomical Distribution and Variations across the Reproductive Cycle of Aromatase Activity and Aromatase-Immunoreactive Cells in the Pied Flycatcher (Ficedula hypoleuca)

The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present.     

Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells

We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca²⁺ concentration and membrane potential revealed coincident photogeneration of Ca²⁺ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca²⁺ which acts on Ca²⁺ activated K⁺ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.     

Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S. cerevisiae: effect of pump overproduction and carbon source

The potentiometric fluorescence probe diS-C3(3) is expelled from S. cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance. The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates. The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained. Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Deltapsi drop in the late exponential and diauxic growth phase, indicating lowered activity of H+ -ATPase. Suppression of activity of both ABC pumps and H+ -ATPase obviously signifies cell transition to an energy-saving mode. Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps--a strong dependence of pump activity on the type of the carbon source.     

Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis

Background

We have recently shown that curcumin (a diferuloylmethane) inhibits growth and induces apoptosis, and also demonstrated that TRAIL induces apoptosis by binding to specific cell surface death receptors in prostate cancer cells. The objectives of this paper were to investigate the molecular mechanisms by which curcumin enhanced the apoptosis-inducing potential of TRAIL in prostate cancer cells.

Results

Curcumin enhanced the apoptosis-inducing potential of TRAIL in androgen-unresponsive PC-3 cells and sensitized androgen-responsive TRAIL-resistant LNCaP cells. Curcumin inhibited the expressions of Bcl-2, Bcl-X_L, survivin and XIAP, and induced the expressions Bax, Bak, PUMA, Bim, and Noxa and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) in both cell lines. Overexpression of dominant negative FADD inhibited the interactive effects of curcumin and TRAIL on apoptosis. Treatment of these cells with curcumin resulted in activation of caspase-3, and caspase-9, and drop in mitochondrial membrane potential, and these events were further enhanced when combined with TRAIL. Curcumin inhibited capillary tube formation and migration of HUVEC cells and these effects were further enhanced in the presence of MEK1/2 inhibitor PD98059.

Conclusion

The ability of curcumin to inhibit capillary tube formation and cell migration, and enhance the therapeutic potential of TRAIL suggests that curcumin alone or in combination with TRAIL can be used for prostate cancer prevention and/or therapy.     

Electrical changes in isolated rat jejunum induced by hypertonicity

Mucosal hypertonicity produces a drop of transepithelial potential difference in isolated rat jejunum with a half time of about 15 s. The same effect is obtained when the osmorality of both bathing solutions is raised simultaneously. Serosal hypertonicity produces an increase of transepithelial potential difference an order of magnitude lower than the drop produced by mucosal hypertonicity. The change in the short circuit current parallels the one in potential difference.When the transepithelial potential difference is varied by adding different concentrations of glucose to the bathing media, the potential drop induced by mucosal hypertonicity is linearly related to the magnitude of the transepithelial potential difference just before the increase in osmolarity.The drop of potential can be explained by a decrease of the electrical resistance of the extracellular shunt pathway due to opening of the tight junctions. The results can be accounted for in terms of an equivalent electrical circuit proposed for small intestine. Using this equivalent circuit model it is possible to obtain estimates of the values of the diffusion potential and the salt gradient across the tight junction.     

Retroactive pathway involving mitochondria in electroloaded cytochrome c-induced apoptosis. Protective properties of Bcl-2 and Bcl-XL

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Deltapsim) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Deltapsim drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.     

Influx流式仪分选肺腺癌A549细胞SP亚群的条件优化

目的：通过分选肺腺癌A549细胞的侧群细胞（side population cells,SP）,了解影响Influx分选SP的各种因素,为成功分选其他肿瘤细胞的SP亚群提供方法参考.方法：（1）细胞的制备：取对数生长期的细胞,制备单细胞悬液,用Hoechst33342及PI标记染色,同时设计维拉帕米对照组;（2）最佳分选喷嘴的确定：根据对数生长期的A549细胞处于不同的分裂时象的细胞大小,预实验确定最佳分选喷嘴;（3）光信号的调试：用RainbowBeads优化670/30[355]、460/50[355]荧光信号和FSC散射光信号,使光信号最强且变异系数（coefficient ofvariation,CV）最小.（4）最佳液滴延迟的确定：调节振荡频率（frequency）找出最佳液流断点位置,调节振幅（ampitude）优化侧液流信号直到两侧液流信号最稳定,Delay Calculator自动计算液滴延迟,根据Accudrop Beads的分选效果确定最佳液滴延迟;（5）其他指标的调整：调节液滴的充电电压（drop charge）和分选装置的X、Y轴坐标,使测试分选液滴准确达到分选装置中;设置分选参数,分选目的细胞群.结果：同一批次处理的A549肺腺癌细胞,100 μm的喷嘴为分选A549肺腺癌SP亚群的最佳喷嘴;CV值校正后SP比例为19.7％,提高了6％;液滴延迟校正后,其分选效率（efficiency）提高了15％,分选纯度（purity）提高了23％;10 μg/mL的Hoechst3342染色,其SP比例是5μg/mL染色的5倍以上;细胞浓度越稀,分选效率和分选纯度相应提高;活细胞比例越高,SP比例越高.结论：合适的喷嘴大小,是保证高分选效率和高分选纯度的基础;仪器较小的CV值和较精确的液滴延迟、合理的Hoechst33342染料浓度、较高的细胞存活率和适中的细胞浓度是保证高分选效率和高分选纯度的的关键;通过平衡上述影响分选的因素才可能得到最佳的分选效果.