Calcein Uptake Research Articles

Quantitative Study of Electroporation-Mediated Molecular Uptake and Cell Viability

Electroporation’s use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1–3.3 kV/cm), pulse length (0.05–20 ms), number of pulses (1–10), calcein concentration (10–100 μM), and cell concentration (0.6–23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation’s effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.

Increase in Doxorubicin Cytotoxicity by Inhibition of P-glycoprotein Activity with Lomerizine.

Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR (multidrug resistance)1 gene encoding the transmembrane efflux pump, P-glycoprotein (P-gp). Calcium channel blockers such as verapamil, nifedipine and nicardipine have been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether a new calcium channel blocker, lomerizine, influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil, and even more markedly, lomerizine, increased cellular uptake of calcein transported by P-gp in a P-gp-expressing erythroleukemia cell line, K562-Dox. Ten microM of lomerizine reduced the IC50 of doxorubicin in the K562-Dox from 60000 ng/ml to 800 ng/ml, whereas the IC50 of doxorubicin in the K562 subline was only marginally affected by these drugs. Lomerizine showed greater reduction in P-gp efflux than verapamil at an equimolar concentration. These results suggest that lomerizine has the clinical potential to reverse tumor MDR involving the efflux protein P-gp.

GFLV replication in electroporated grapevine protoplasts

Grapevine fanleaf virus (GFLV), responsible for the economically important court-noué disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 μg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

Effects of static pressure on ultrasound-mediated transdermal delivery of tagging compounds into migratory fish

Research is currently being conducted with the US Fish and Wildlife Service investigating the use of ultrasound to enhance the transdermal delivery of tagging compounds into migratory fish. There is a need for noninvasive, low-cost, and efficient methods of tagging large numbers of fish in order to follow their migratory patterns. Current methods require long periods of immersion that can create health problems and are limited in the amount of uptake of the compound. Previously reported research [J. A. Clark et al., J. Acoust. Soc. Am. 101, 3138(A) (1997)] showed cavitation levels of ultrasound (2–3 W/cm2) at 71 kHz significantly enhanced the transdermal delivery of the tagging compound calcein (fluorexon) into rainbow trout (Salmo gairdneri) yoke-sac larvae. However, attempts to repeat the experiment at higher altitudes revealed that static pressure critically affected the results. A further investigation into the effect of static pressure on the enhanced delivery of calcein by ultrasound into rainbow trout is reported here. A system for generating controlled doses of cavitation level ultrasound at various levels of ambient pressure will be described. The effect of static pressure on calcein uptake will be shown via fluorescent microscopy.

Sonoluminescence as an indicator of cell-membrane disruption by acoustic cavitation

Acoustic cavitation has been shown to reversibly disrupt cell membranes and thereby transport molecules into viable cells. The hypothesis was that the light emitted during inertial cavitation, i.e., sonoluminescence, correlates with these acoustic bioeffects. In this study, sonoluminescence was measured using a photomultiplier tube mounted at the base of an opaque cavitation chamber. The bioeffects measured on DU 145 prostate cancer cells placed in the cavitation field included the number of molecules delivered into each cell and overall cell viability. A 24-kHz cylindrical piezoelectric transducer was used in these experiments to generate cavitation over a range of pressure levels, pulse lengths, and total exposure times. The results show that cells were reversibly disrupted, yielding extensive uptake of calcein by some cells, little uptake by other cells, and loss of viability for a third population of cells. Correlation with sonoluminescence indicated that light emission correlated with these bioeffects.

Increase in doxorubicin cytotoxicity by carvedilol inhibition of P-glycoprotein activity.

Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR1 (multidrug resistance) gene encoding for the transmembrane efflux pump, P-glycoprotein (P-gp). The calcium channel blocker verapamil has been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether the new antihypertensive drug carvedilol influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil (10 micromol/L), and even more markedly, carvedilol (10 micromol/L) increased cellular uptake of P-gp-transported calcein of a P-gp-expressing breast cancer cell line (Hs578T-Dox). In the subline (Hs578T) not expressing P-gp, no effects of carvedilol or verapamil on calcein uptake were seen. Carvedilol and verapamil (10 micromol/L) reduced the LD50 (dose which results in the death of half the number of cells) of the Hs578T-Dox subline from 200 mg/L to approx. 10 mg/L Dox, whereas the LD50 of the Hs578T subline was only marginally affected. Carvedilol (10 micromol/L) reduced P-gp activity approximately twice as effectively as verapamil at an equimolar concentration. Carvedilol did not affect pyrogallol cytotoxicity and pyrogallol was without effect on calcein accumulation of the Hs578T-Dox cell line, indicating the lack of antioxidative properties affecting P-gp activity and associated toxicity of the drug. The results suggest that carvedilol has the clinical potential to reverse tumour MDR involving the efflux protein P-gp.

Extracellular Formation of Body and Tunic Spicules in the New Zealand Solitary Ascidian Pyura pachydermatina (Urochordata, Ascidiacea)

AbstractThe New Zealand ascidian Pyura pachydermatina has a 7–10 cm long body at the end of a stalk up to 1 m long and 1–2 cm in diameter. Two different spicule types are present: dumbbell‐shaped spicules of calcite in the fibrous tunic that covers the body and stalk, and antler‐shaped spicules of amorphous calcium carbonate in the soft body tissues. Both types form extracellularly within a closed compartment surrounded by an epithelium of sclerocytes. In adults the tunic spicules form in 2–3 weeks in the lumen of the tunic blood vessels, as determined by calcein uptake studies. They add mineral only while surrounded by the sclerocyte epithelium, which is anchored to the vessel wall. Ultimately the sclerocytes rupture at one or more leading points on the spicule. The blood vessel epithelium also becomes very thin at these points and either ruptures or the cells separate. allowing the spicules to migrate out into the tunic. The sclerocytes degenerate and the blood vessel closes behind the migrating spicule, thus maintaining the vessel's integrity. Tunic spicules accumulate in the subcuticular region of the stalk, but the outermost layer of tunic covering the body is periodically sloughed off along with some spicules. This gives the “neck” between body and stalk a flexibility that allows it to orient to currents, and prevents an accumulation of epizoic organisms on the body. The antler spicules form within blood sinuses of the body tissues. The mineral and organic material are arranged in concentric layers. In the branchial sac, oral tentacles, gut and endostyle, where antler spicules occur most densely, the branches interlock, providing support to the soft tissues. They are of many sizes and apparently remain where they form, increasing in number and size throughout the animal's lifespan.

Rates of systemic degradation and reticuloendothelial system uptake of calcein in the dipalmitoylphosphatidylcholine liposomes with soybean-derived sterols in mice.

The systemic degradation and the reticuloendothelial system (RES) uptake of calcein entrapped in dipalmitoylphosphatidylcholine (DPPC) liposomes with soybean-derived sterols (SS) were examined after intravenous administration to mice by measuring the free and liposomal calcein levels in the blood. The results indicate that the rates of systemic degradation and the RES uptake of liposomes decrease with the addition of SS in DPPC liposomes since the SS has the ability to stabilize the liposomes. The rate of uptake by RES is larger than the rate of systemic degradation. The rate of leakage of calcein from liposomes by incubation in plasma in vitro is almost the same as that of systemic degradation in vivo.

Calcein accumulation as a fluorometric functional assay of the multidrug transporter

Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein - Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493–21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.

Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.

Mineralization of adult mouse bone marrow in vitro.

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.