Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.     

Increased mitochondrial uptake of rhodamine 123 by CDDP treatment.

Rhodamine 123 (R 123) is a positively charged dye at physiological pH that accumulates specifically in the mitochondria of living cells without cytotoxic effect. In the present study, the uptake of R 123 by EL-4 lymphoma cells in culture with anticancer agents was measured by flow cytometry. Changes in R 123 uptake during the cultivation period were compared with cell distribution at different phases of the cell cycle. According to the increase in the proportion of S phase cells, mitochondrial synthesis increased, giving rise to a maximal fluorescence intensity of about 1.3-fold. Synchronous cultures showed the same relationship between increased mitochondrial uptake of R 123 and the S phase fraction as was observed in normal cultures. After treatment with 10(-3) M 5-fluorouracil (5-FU) for 1 h, EL-4 cells showed an increased binding of R 123 per cell followed by an accumulation of early S phase cells transiently. However, uptake of R 123 decreased 24 h later. On the contrary, after treatment with 10 micrograms/ml of cis-diamminedichloroplatinum (CDDP), a G2 + M block was observed from 12 h of reseeding and accumulation of the G2 + M cells continued. In this case, high uptake of R 123 continued during the observation period. From these results, mitochondrial synthesis seemed to increase according to the increment in proportion of S phase when the acceleration of the cell cycle turnover was augmented or the cycle was blocked in S phase by 5-FU. CDDP inhibited the cell division at G2 + M phase and caused increased R 123 fluorescence per cell. The stainability of R 123 may indicate the activity of cell division and may be a good way of evaluating the efficacy of antitumor drugs on the cells.     

The influence of acute hypoxia on the functional and morphological state of the black scorpionfish red blood cells

The investigation of the mechanisms of red blood cell steadiness to the oxygen lack in tolerant teleosts is of current scientific interest. Black scorpionfish, Scorpaena porcus L., is a widespread benthal species in the Black Sea and is highly resistant to hypoxic influence. The morphological state of black scorpionfish red blood cells under acute hypoxia was assessed using DNA-binding dye SYBR Green I and fluorescent microscopy. Changes in membrane potential of mitochondria and functional activity of cells were determined by rhodamine 123 (R123) and fluorescein diacetate (FDA) fluorescence. Oxygen deficiency leads to bidirectional changes in volume of erythrocytes and their nuclei. Between 0.57 and 1.76 mg О₂ l^?1, both parameters increased on 3–12 and 7–21%, respectively. At 1.76–4.03, cells shrank on 1.5–6.0% and nucleus size decreased on 1.5–3%. Acute hypoxia induced a significant increase of R123 (12–60%) and FDA (30–184%) fluorescence. These reactions are caused by a probable decrease in erythrocyte membrane permeability.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Simultaneous use of rhodamine 123, phycoerythrin, Texas red, and allophycocyanin for the isolation of human hematopoietic progenitor cells

The supravital, mitochondrial specific dye Rhodamine 123 (R123) was used in conjunction with three monoclonal antibodies to isolate a population of human bone marrow (BM) cells enriched for hematopoietic progenitor cells. BM cells stained with phycoerythrin-HLA-DR, Texas red-CD34, allophycocyanin-CD15, and R123 were fractionated using four-color immunofluorescence cell sorting. Cells expressing CD34 but not HLA-DR and CD15 (CD34+ HLA-DR- CD15-) were subdivided according to their reactivity with R123 into quiescent, R123 dull (R+) or cycling, R123 bright (R++) subpopulations. Morphological analysis and hematopoietic progenitor cell assays indicated that CD34+ HLA-DR- CD15- R+ cells contained larger numbers of blast cells and colony forming units than CD34+ HLA-DR- CD15- R++ cells. The flow cytometer settings used to accommodate the detection of the R123 fluorescence in combination with that of three other fluorochromes are described.     

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10–20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo. J. Cell. Physiol. 176:138–149, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Capsaicin-Induced Changes in the Cytosolic Calcium Level and Mitochondrial Membrane Potential

Simultaneous video-microfluorimetry allows experimenters to monitor calcium signals in the cytosol, as well as changes in the membrane potential of the mitochondria, in living cells loaded with both fura2 and rhodamine123 (rhod123). Capsaicin-evoked responses of cultured sensory neurons and transfected HT1080 cells are described below. Polymodal nociceptors [1] or other cells expressing TRPV1 receptors respond to capsaicin application with a rise in the cytosolic calcium level ([Ca²⁺]_c), reaching eventually toxic levels. Capsaicin induces selective permanent morphological changes of the mitochondria before any loss of small cells (type B) in the sensory ganglia can be detected [3]. An unknown link between changes in the mitochondria and cell loss can be investigated by combined functional examination of capsaicin-induced [Ca2+]_c changes and reactions of the mitochondria. In most tests, the capsaicin-induced [Ca²⁺]_c elevation occurred before the rising phase of rhod123 waves. Cellular reactions were either transient or sustained (lasting over hundreds of seconds). A transient or a sustained nature of the reactions was slightly concentration-dependent. Fluorescence of the cells changed in complicated ways during repeated tests. Moderate but permanent changes of the cellular responsiveness suggest mild injury, which might be involved in cellular desensitization.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 82–93, January–February, 2005.     

Mitochondrial behaviour during the yeast-hypha transition of Candida albicans

Yeast cells of Candida albicans were brought to germ tube formation and hyphal growth in liquid synthetic medium. The behaviour of mitochondria and mitochondrial nucleoids (mt-nucleoids) during morphological conversion was examined by fluorescence staining with 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) and 4',6-diamidino-2-phenylindole (DAPI). Parent yeast cells possessed one or very few branched giant mitochondria which were stained intensely with DASPMI. When a germ tube emerged from the parent cell, one end of a giant mitochondrion extended into the germ tube and developed into the elongated form. In mycelia, apical hyphal cells contained giant mitochondria, whereas older hyphal compartments near the parent cells were vacuolated and possessed small, peripherally located mitochondria. The vacuolated hyphal compartments resynthesized cytoplasm before producing branches and contained giant mitochondria. The cytological model for germ tube formation and hyphal growth proposed by Gow and Gooday (1984) is discussed.     

Thapsigargin induces mitochondrial dysfunction and apoptosis in the mastocytoma P815 cell line and in mouse thymocytes

Thapsigargin is a plant-derived inhibitor of the endoplasmic reticulum Ca(2+)-ATPase.Treatment with thapsigargin leads to a rapid, large and prolonged increase in the intracellular calcium ion concentration ([Ca(2+)](i)). Previously thapsigargin has been shown to inhibit proliferation and induce apoptosis. Here we report the results of thapsigargin treatment in thymocytes harvested from 10-day-old mice and in the P815 mastocytoma cell line. In thapsigargin-treated cells we observed enlarged mitochondria with disrupted cristae structure. These mitochondria closely resembled those observed after the induction of phase transition. To determine if the mitochondria were functioning normally the cells were stained with rhodamine 123 (R123) and analysed with flow cytometry. After thapsigargin treatment the R123 staining decreased, indicative of a loss of mitochondrial membrane potential. Furthermore intracellular ATP concentrations were also found to be reduced in cells treated with thapsigargin. Taken together these results indicate an increase in the [Ca(2+)](i) caused by thapsigargin treatment results in dysfunctional mitochondria and reduced ATP. We propose that this decrease in the concentration of ATP provokes the onset of thapsigargin-induced apoptosis. To investigate the effect of thapsigargin treatment on the cell cycle, rapidly cycling P815 cells were sorted into populations enriched for either G(0)/G(1) or S/G(2)/M phases, and these populations were then treated with thapsigargin. Thapsigargin treatment induced a cell cycle block before S phase. We propose that the block in the cell cycle induced by thapsigargin was a result of the decreased intracellular ATP concentration interfering with the energy requiring processes of DNA replication. The block could also be related to the high intracellular calcium ion concentration that would interfere with the subtle calcium transients involved in the cell's preparations for replication and mitosis. Apoptosis occurred to an equal extent in both populations of cells.     

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well.     

Swelling-activated chloride channels in multidrug-sensitive and - resistant cells

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug- efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug- sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively. gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV. Cl- channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells. gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM). None of these blockers inhibited R123 efflux. NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered. Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling- activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Mitochondrial membrane potential regulation is independent of c-fos expression.

Tumour cells contain mitochondria with elevated membrane potentials compared with normal cells, and thus this feature provides a selective target for destroying tumour cells. To improve mitochondrial-based therapies, a better understanding of the factors involved in regulating mitochondria are required. Since v-fos overexpression has been shown to elevate mitochondrial membrane potentials in rat fibroblasts, we investigated whether the human homologue, c-fos, was also capable of regulating the mitochondrial membrane potential in cells. Rat fibroblasts transfected with the c-fos gene did not accumulate more rhodamine 123 (Rh123) nor did they retain this Rh123 for extended periods of time compared with their parental line. Moreover, there was no difference in survival following dequalinium chloride (Deca) treatment between transfectants and controls. Similarly, reduction of c-fos expression in rat fibroblasts did not significantly alter their mitochondrial membrane potential. In addition, human ovarian carcinoma cells, which overexpress the c-fos gene, did not accumulate more Rh123 nor were they hypersensitive to Deca compared with their parental line. In another human ovarian carcinoma cell line, selection of variants with lower mitochondrial membrane potential did not alter c-fos mRNA or protein levels. These data suggest that alterations in c-fos expression do not regulate the magnitude of the mitochondrial membrane potential.     

Evaluation of mitochondrial content and activity with nonyl-acridine orange and rhodamine 123: flow cytometric analysis and comparison with quantitative morphometry

Mouse fibroblasts 3T3.4E and two cell lines obtained by fusion (3T3.4E cells x normal human keratinocytes), (3T3 x NHK), and (3T3.4E cells x hand wart keratinocytes), (3T3 x HWK), were compared for mitochondrial activity and content between 5 and 20 days of culture, from the 16th to 20th passage, by using Rh 123 and NAO respectively. In 3T3.4E cells both Rh 123 and NAO fluorescence were similar after 5 and 7 days of culture, indicating no modification of mitochondrial activity and content at that time. However, in cells derived from fusion of 3T3 x NHK or 3T3 x HWK, Rh 123 increased from 5 to 20 days whereas NAO fluorescence was maximal at 7 days of culture and then decreased, indicating that their mitochondrial activity differed from that of 3T3.4E cells. No difference was observed between the 16th and 20th passage. Quantitative morphometry and flow cytometry gave good correlations at 7 days of culture for the cell size, estimated either by the cell area or the cell diameter, and for mitochondria content, evaluated either by the number of mitochondria per cell or NAO fluorescence intensity.Abbreviations FCS Fetal Calf Serum - mt DNA mitochondrial DNA - NAO nonyl-acridine orange - PBS Phosphate Buffer Saline - Rh 123 Rhodamine 123 - 3T3 x NHK (3T3.4E cells x normal human keratinocytes) - 3T3 x HWK (3T3.4E cells x hand wart keratinocytes)     

Rhodamine 123 as a vital stain for mitochondria of plant cells

Abstract. Rhodamine 123 (Rh 123), a relatively new mitochondrial marker, little used in the study of plant cells, was tested on excized leaves of Elodea canadensis Michx. and on suspension-cultured cells of Ranunculus serbicus Vis. In both preparations, the dye accumulated rapidly and selectively in the mitochondria whose number, morphology and cell distribution could be easily observed. In the presence of Rh 123, cytoplasmic movements could also be perceived and the spatial arrangement of the mitochondria with respect to that of the auto-fluorescent chloroplasts was studied in connection with a normal or altered cytoskeletal framework. The specific uptake of Rh 123 by the organdies seemed to be potential-dependent since it was influenced by cations, ionophores and inhibitors of electron transport. Short exposures to the stain were practically non-toxic, whereas prolonged treatments (6–20 h) provoked specific alterations in structure of the mitochondria. The data reported here indicate that Rh 123 may be an excellent vital stain to study the morphology, function and dynamics of the mitochondria in living plant cells.     

Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose.

The phosphatase inhibitor okadaic acid was found to induce cell cycle arrest of human myeloid leukemic cell lines HL-60 and U937 in a concentration- and time-dependent manner. Exposure to low concentrations of okadaic acid (2-8nM) for 24-48 hr caused greater than 70% of cells to arrest at G2/M, with up to 40% of the cells arrested in early mitosis. Cell viability decreased rapidly after 48 hr of treatment, and morphological and DNA structure analysis indicated that this was primarily due to the induction of apoptosis. The cells arrested in mitosis by 8 nM okadaic acid could be highly enriched by density gradient centrifugation and underwent apoptosis when further cultured either with or without okadaic acid, indicating that the effects of okadaic acid were irreversible. In contrast to the effects of low concentrations of okadaic acid, high concentrations (500 nM), inhibited proliferation in less than 3 hr. Remarkably, the majority of cells also entered a mitosis-like state characterized by dissolution of the nuclear membrane and condensation and partial separation of chromosomes. However, these cells had a diploid content of DNA, indicating that the cell cycle arrest occurred at G1/S with premature chromosome condensation (PCC), rather than at G2/M. If cells were first blocked at G1/S with hydroxyurea and then treated with okadaic acid, greater than 90% developed PCC in less than 3 hr without replicating their DNA. Caffeine was not able to induce PCC in these cells, either with or without prior inhibition of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ambient particulate matter induces alveolar epithelial cell cycle arrest: role of G1 cyclins

We hypothesized that the ambient air pollution particles (particulate matter; PM) induce cell cycle arrest in alveolar epithelial cells (AEC). Exposure of PM (25microg/cm(2)) to AEC induced cells cycle arrest in G1 phase, inhibited DNA synthesis, blocked cell proliferation and caused decrease in cyclin E, A, D1 and Cyclin E- cyclin-dependent kinase (CDK)-2 kinase activity after 4h. PM induced upregulation of CDK inhibitor, p21 protein and p21 activity in AEC. SiRNAp21 blocked PM-induced downregulation of cyclins and AEC G1 arrest. Accordingly, we provide the evidence that PM induces AEC G1 arrest by altered regulation of G1 cyclins and CDKs.     

Significance of the cytoskeleton for cytoplasmic organization and cell organelle dynamics in epithelial cells of fresh-water sponges

Summary The cytoskeleton in epithelial cells ofSpongilla lacustris is constructed of microtubules radiating from the nuclear region and terminating at the cell periphery as well as microfilaments forming a cortical layer beneath the plasma membrane and distinct fibers in the cytoplasmic matrix. Single frame analysis and in vivo labeling of mitochondria with Rh 123, endosomes or lysosomes with TRITC-BSA, endoplasmic reticulum (ER) with DiOC₆ (3) and dictyosomes with C₆-NBD-ceramide points to the microtubular system as a candidate for controlled cytoplasmic organization and active transport of these cell organelles. In epithelial cells with an intact microtubular system, mitochondria and endosomes or lysosomes show a regular shuttle movement between the nucleus and the cell periphery with a velocity of 1.3–1.4 m/s; the ER forms a more or less dynamic two-dimensional network in the entire cytoplasmic matrix, and dictyosomes are arranged in a ring-like manner around the nucleus. In epithelial cells treated with colchicine or colcemid, mitochondria and endosomes or lysosomes gather in the perinuclear region and become immobile; the ER accumulates near the cell center, whereas most dictyosomes distribute randomly over the whole cytoplasm. Transformation of the microfilament system with cytochalasin D has no influence on cell organelle distribution and dynamics but impedes cell locomotion and cell surface activities.Abbreviations BSA bovine serum albumin - C₆-NBD-ceramide 6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)caproyl]sphingosine - DiOC₆(3) 3,3-dihydroxyloxacarbocyanine jodide - DMSO dimethylsulfoxide - EGTA ethylenediaminetetraacetic acid - GTX glycerol-Triton-X-100 - PBS phosphate buffered saline - PEG polyethylene glycol - PIPES 1,4-piperazine-N,N-bis-(2-ethanesulfomc) acid - Rh 123 rhodamine 123 - TRITC tetramethylrhodamine isothiocyanate     

Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange

Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.