Use of 1,4 diacetoxy-2,3 dicyanobenzol in microspectrofluorometry for determining the intracellular pH of isolated living cells

Microspectrofluorometry allows to obtain the fluorescence spectrum of an isolated living cell. When cells are preincubated with 1,4 diacetoxy-2,3 dicyanobenzol the cellular fluorescence spectrum can be resolved in its components i.e. the characteristic fluorescence spectrum of each ionized forms of the probe and the intrinsic cell fluorescence spectrum due to NAD(P)H. This allows the determination of the intracellular pH with good accuracy. Furthermore, comparison between the intensity of the intrinsic cell fluorescence and the probe fluorescence intensity offers us an opportunity to monitor the intracellular amount of the drug.     

Manipulation of living cells by using PC-controlled micro-pattern projection system

In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we developed a novel technique to capture the living cells on a culture substrate by irradiating light in a multiple manner. In clear contrast to the conventional cell patterning using the previously patterned substrate, the cell-retaining area can be defined even after cell seeding, and the captured cells can continue to grow freely beyond the defined area afterwards. After the light irradiation in arbitrary micropattern by using a newly developed apparatus and the process to remove non-captured cells including EDTA treatment, the highly contrasted cell patterns were formed with the precision of single cell size. Moreover, it was determined that the cell capturing arose just after light irradiation and diminished gradually in a time scale of 10h. It was confirmed that the cells maintained their viability well after the manipulation process including photo-induced cell capturing.     

Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into living cells

We have developed a novel method for introducing exogenous macromolecules from solution into the cytoplasm of living amoebae of the cellular slime mold Dictyostelium discoideum and have used it to measure the cytoplasmic pH of these cells. Amoebae (strain NC-4) were loaded with fluorescein-labelled dextran by sonication in a solution containing 17 mM phosphate buffer, 1 mM CaCl2, and 10 mg/ml of fluorescein-labelled dextran, pH 6.1. The recovery of living cells was approximately 40% after sonication and washing. A significant fraction (10%) of the recovered cells were loaded and contained 10(5) to 10(7) molecules of fluorescein-labelled dextran per cell as assessed by flow cytometry. The cells loaded by sonication appeared both viable and healthy, since they exhibited normal morphology and locomotion, could differentiate to form mature fruiting bodies, could chemotax in a gradient of extracellular cAMP, and could endocytose latex microspheres. The pH of single cells was estimated by using flow cytometry to measure the fluorescence ratio (fluorescein/rhodamine) in cells loaded with a mixture of the two fluorochrome-labelled dextrans. The fluorescence ratios were calibrated in situ with the flow cytometer after treatment of the cells with either weak acid or weak base to clamp the internal pH at known values. The intracellular pH measured in cells loaded with dextran in a simple salt solution was 5.9. The intracellular pH measured in cells loaded with dextran in the same solution supplemented with amino acids and glucose was 6.7. The novel sonication loading technique described may have general utility for loading diverse types of macromolecules into suspensions of living cells.     

Rapid method for measuring intracellular pH in vivo.

Intracellular pH (pHi) was simultaneously measured in 6 normal tissues and a malignant tumour of rats by a rapid triple isotope technique, based on the in vivo distribution of 5,5-dimethyl-2,4-oxazolidinedione-2-14C (DMO), tritiated water and sodium chloride-36. Results compared favourably with pH measured directly in the same rat by capillary glass electrode, and with values of other workers for pHi in rat tissues. Mean pHi of normal tissues was close to pH 7, and in each organ there was a linear relationship between pHi and extracellular pH (pHe) over the normal range of pHe encountered (pH 6.9-7.6). Organ pHi altered in response to administration of NH4Cl or NaHCO3 to the host.     

Schistosoma japonicum: a method for transformation by electroporation

Despite of our knowledge of genetic make up of schistosomes, a number of genes have not been characterized largely due to lack of effective transformation protocols. Here we present electroporation as a strategy for effective introduction of plasmids DNA into schistosomula and adults. Using plasmids of pEGFP-C1 as an expression vector, we first verified that the CMV promoter could direct EGFP to express in primary culture cells from Schistosoma japonicum. Subsequently, the plasmids were introduced into schistosomula and adults by electroporation and EGFP expression was demonstrated using molecular and microscopical methods. Our findings indicate that electroporation is an effective method for transformation of S. japonicum.     

Resistance induction in barley coleoptile cells by intracellular pH decline

Cytoplasmic acidification in suspension-cultured plant cells has been characterized as a common intracellular response of some kinds of plant cells to elicitors. Expression of various defense genes in these cells has been increased by the cytoplasmic acidification itself without treatment by elicitors. It is not evident, however, whether or not cells with acidified cytoplasm actually exhibit resistance to the pathogen because of the lack of an adequate infection system between cultured plant cells and some pathogens. Using barley coleoptiles rather than suspension-cultured cells, we demonstrated both detection of cellular pH decline and increased resistance to Blumeria graminis. The cytoplasmic pH of barley coleoptile cells floated on 1 mM citrate buffer (CB), pH 4.0, became 0.5 unit lower than that of cells floated on 1 mM CB, pH 8.0, within 30 min after treatment. The penetration efficiency of B. graminis into the coleoptile was decreased in a pH-dependent manner; that is, when the coleoptiles were floated on 1 mM CB, pH 8.0, the penetration efficiency of the fungi was about 80%. In contrast, when the coleoptiles were floated on acidic buffers, the penetration efficiency decreased in parallel the decline of pH and the penetration efficiency reached 0% when coleoptiles were floated on 1 mM CB, pH 4.0. Morphogenesis of appressoria on the coleoptiles floating on CB was not influenced. The lowered penetration efficiency at lower pH was partially cancelled when the barley coleoptiles were irradiated with UV for 5 min prior to B. graminis inoculation. These findings suggest that the decline in cytoplasmic pH in barley coleoptile cells increases resistance to the pathogenic fungus B. graminis.     

Brilliant Blue as an alternative dye to Fast Green for in ovo electroporation

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post-injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co-electroporation.     

A rapid method for measuring intracellular pH using BCECF-AM

A rapid intracellular pH (pH(i)) measurement method based on initial rate of increase of fluorescence ratio of 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein upon dye addition to a cell suspension in growth medium is reported. A dye transport model that describes dye concentration and fluorescence values in intracellular and extracellular spaces provides the mathematical basis for the approach. Experimental results of ammonium chloride challenge response of the two suspension cells, Spodoptera frugiperda and Chinese hamster ovary (CHO) cells, successfully compared with results obtained using traditional perfusion method. Since the cell suspension does not require any preparation, measurement of pH(i) can be completed in about 1 min minimizing any potential errors due to dye leakage.     

Improved intracellular delivery of oligonucleotides by square wave electroporation

Prior studies have shown that electroporation is a simple and effective method for the introduction of oligonucleotides (ODN) into cells. In ex vivo bone marrow purging models, electroporation of ODN into cells has been associated with selective killing of human neoplastic cells while sparing hematopoietic stem cells. Prior studies used conventional electroporation methods (i.e., exponential decay) to introduce ODN into cells. Square wave electroporation allows the delivery of a more defined and regulated electrical pulse and is associated with high transfection efficiencies in a variety of systems. The current study was undertaken to determine whether square wave electroporation was more effective than exponential decay electroporation for the delivery of ODN into hematopoietic cells. Using fluorescein-tagged ODN and K562, chronic myelogenous leukemia (CML) cells, higher transfection rates were observed after square wave electroporation. In addition, c-myc antisense ODN were more effective in reducing c-myc protein when introduced by square wave electroporation, as compared with introduction by exponential decay electroporation. Square wave electroporation is thus identified as the optimal method for delivering ODN into hematopoietic cells.     

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).     

An alternative method for a fast separation of phosphotyrosine.

A simple and rapid procedure is described for fully separating phosphotyrosine from phosphoserine and phosphothreonine through one-dimensional thin-layer chromatography. The migration properties of these phosphoamino acids are compared with those of CMP, UMP, ATP, ribose phosphate, and inorganic orthophosphate, considered the most frequent comigrating products derived from 32P-labeling experiments. We demonstrate that Rf values for the three phosphoamino acids differ from those displayed by the mentioned contaminating compounds. One of the most relevant advantages of this procedure is that a complete separation of phosphotyrosine can be achieved in only 90 min.     

Chemoporation using saponins or cholates: an alternative method for transformation of bacterial cells

A new method for fast transformation of competent bacterial cells has been developed. The transformation is induced with cholic acid analogues or saponins which cause reversible disruption of the bacterial membrane. This method shortens the time of transformation without significant loss of transformation efficiency in comparison to heat shock method and is the first reported chemically-induced transformation. New data about interactions between cholates and biomembranes is revealed that may contribute to better understanding of bacterial transformation.