The influence of products from electrolysis reactions occurring during high-voltage electric impulses on fluorescent molecules

Fluorescenciniai dažai dažnai naudojami norint studijuoti ląstelių membranos elektropermeabilizacijos procesą. Kuomet elektrolitų tirpale yra taikomas aukštos įtampos impulsas, įvyksta daug įvairių elektrolizės reakcijų. Iš elektrodų išsiskyrę metalų jonai gali reaguoti su fluorescenciniais dažais ir slopinti jų fluorescenciją. Būtent tai gali turėti įtakos siekiant įvertinti elektropermeabilizacijos efektyvumą. Šiame darbe, terpės, veiktos aukštos įtampos impulsu ir Al3+, Fe2+, Fe3+, Cr6+, Ni2+ ir Mn7+ jonais, įtaka fluorescuojančių molekulių fluorescencijai, įskaitant fluorescencinį indikatorių vandenilio peroksidui, įvairiose terpėse buvo tiriama. Ląstelių kultūros terpė susidedanti iš Dalbeko modifikuotos Iglo terpės papildytos su 9 % jaučio vaisiaus serumu ir 1 % L-glutamino tirpalu (visos iš Sigma-Aldrich Chemie, Steinheim, Vokietija). 50 l mitybinės terpės arba fluorescencinio dažo tirpalo buvo paveikta 0,1–2 ms trukmės ir 0,2–2,4 kV/cm amplitudės kvadratiniu elektriniu impulsu. Buvo tiriama kalceino, mezo-tetrakio (4-sulfonatofenilo) porfirino (TPPS4), doksorubicino (adriamicino) ir H2O2 indikatoriaus Amplekso raudonojo fluorescencija. Fluorescencija buvo matuojama kambario temperatūroje, naudojant Tecan spektrofluorimetrą (Tecan Group, Mannedorf, Šveicarija). Terpė veikta 1,2 kV/cm amplitudės, 500 ir 2000 µs trukmės elektriniu impulsu beveik visiškai nuslopino kalceino fluorescenciją. Didėjant metalo jonų koncentracijai tirpale kalceino, TPPS4, adriamicino ir Amplekso raudonojo fluorescencijos intensyvumas sumažėjo. Pavyzdžiui, 1mM Fe3+ arba Ni2+ jonai nuslopino 15 ir 79 % kalceino fluorescenciją. 1 mM koncentracijos Fe3+ visiškai nuslopino porfirino-sulfonato fluorescenciją, 30 % adriamicino fluorescenciją ir 30–50 % Amplekso raudonojo fluorescenciją. Galima daryti tokią išvadą, kad ląstelių kultūros terpė, paveikta aukštosios įtampos impulsu, slopina įvairių fluorescencinių molekulių fluorescenciją, o daugiausia dėl to, kad iš nerūdijančio plieno ir aliuminio lydinio elektrodų išsiskyrė metalų jonai. Visi šiame moksliniame darbe atlikti tyrimai gali būti naudingi optimizuojant elektroporacijos metodą, bei jį įdiegiant klinikinėje praktikoje.To study cell membrane electropermeabilization, fluorescent dyes are often used. When a high-voltage pulse is applied to the electrolyte solution, a variety of electrolysis reactions occur at the metal-electrolyte interfaces. Metal ions released from the electrodes can react with the fluorescent dyes and quench their fluorescence. This may have an impact when estimating the efficiency of electropermeabilization. In this study, the influence of the medium treated by high-voltage pulse and Al3+, Fe2+, Fe3+, Cr6+, Ni2+ and Mn7+ ions on the fluorescence of fluorescent tracer molecules, including fluorescent indicator for hydrogen peroxide, in various solutions was studied. Cell culture medium consisted of Dulbecco‘s modified Eagle‘s medium supplemented with 9 % fetal bovine serum and 1 % L-glutamine solution (all Sigma–Aldrich Chemie, Steinheim, Germany). 50 µl of the culture medium or a solution of a fluorescent dye was treated with a square-wave electric pulse with the duration of 0,1–2 ms and the amplitude 0,2–2,4 kV/cm. The fluorescence of calcein, meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS4), doxorubicin (Adriamycin) and H2O2 indicator Amplex Red was studied. The fluorescence was measured at room temperature using Tecan spectrofluorimeter (Tecan Group, Männedorf, Switzerland). The medium treated by the electric pulse with the amplitude of 1,2 kV/cm and the duration of 500 and 2000 µs almost completely quenched calcein fluorescence. With increasing the concentration of the metal ions in solution the intensity of the fluorescence of calcein, TPPS4, Adriamycin and Amplex Red decreased. For example, 1 mM of Fe3+ or Ni2+ ions suppressed fluorescence of calcein by 15 and 79 % respectively. Fe3+ at the concentration of 1 mM totally suppressed the fluorescence of porphyrin-sulphonate, by 30 % – the fluorescence of Adriamycin, and by 30–50 % the fluorescence of Amplex Red. It can be concluded that the cell culture medium pretreated by high-voltage pulse quenches the fluorescence of various fluorescent molecules, mainly due to the metal ions released from the stainless-steel and aluminum electrodes. The results of the present work can be useful for optimizing the electroporation method as well as for introducing it to medicine or industry.Gamtos mokslų fakultetasVytauto Didžiojo universiteta

Chitin extraction and chitosan production from Chilopoda (Scolopendra cingulata) with identification of physicochemical properties

WOS: 000358218500009The Chilopoda is found in a broad array of terrestrial habitats from tropical rain forests to deserts and there are more than 8000 species worldwide. Chitin and chitosan were obtained from Scolopendra cingulata for the first time in this study and physico-chemically characterized. The dry weight chitin content of S. cingulata was found to be 8% of body weight, and it produced chitosan yields of 66%. Analyses results revealed that Chilopoda chitin was in the alpha-form. The chitosan derived from Chilopoda chitin has a low molecular weight (2.278 kDa). The surface morphology of chitin from S. cingulata consists of dispersed nanopores and nanofibres

Study of the influence of iron ions on the fluorescence of calcein and calcein blue

The process of a temporal increase of the cell membrane permeability occurring due to the action of the pulses of strong electric field (up to 300 kV/cm) is called cell electroporation. This process is widely used in cell biology, biotechnology, and medicine [1]. When high–voltage pulse is applied to the electrolyte solution the oxidation of metal ions of the anode occurs besides other electrolysis reactions. As a result of this, the dissolution of the anode takes place [2]. When metal ions released from the electrodes, they reacts with fluorescent molecules and reduce the intensity of their fluorescence [3, 4]. Stainless– steel is one of the most popular materials utilized for electrodes, which are used to electroporate the cells. In these conditions, iron ions (Fe2+ and Fe3+) are released from the anode under the action of high–voltage electric pulses. In the present research, the impacts of iron ions on the fluorescence of calcein and calcein blue in three different media – distilled water, 0.9% NaCl and Dulbecco\'s Modified Eagle Medium (DMEM) – have been studied. The most suppressed fluorescence was found to be in 0.9% NaCl media and the least suppresed in DMEM media. For example, in 0.9% NaCl media only 0.1 mM of Fe2+/Fe3+ was required to fully suppress the fluorescence of calcein, while in the case of calcein blue, much higher concentration of iron ions - 1 mM - was required to get the same effect. The results of this reasearch can be useful when estimating the efficiency of cell electropermeabilizationBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Influence of iron ions on the fluorescence of calcein and calcein blue

Online ISSN: 2335-8718Cell electroporation – a temporal increase of the cell membrane permeability occurring due to the action of the pulses of strong electric field (up to 300 kV/cm) – is widely used in cell biology, biotechnology, and medicine. When a high–voltage is applied to the electrolyte solution, besides other electrolysis reactions, the oxidation of the metal ions of the anode occurs. As a result of this, the dissolution of the anode takes place. The metal ions, which are released from the electrodes, can react with fluorescent molecules and decrease the intensity of their fluorescence. One of the most popular materials utilized for electrodes, which are used to electroporate the cells, is stainless–steel. In such a case, iron ions (Fe2+ and Fe3+) are released from the anode under the action of high–voltage electric pulses. In the present work, the effects of iron ions on the fluorescence of calcein and calcein blue in three different media – distilled water, 0.9% NaCl, and Dulbecco's Modified Eagle Medium (DMEM) – have been studied. The fluorescence was found to be the most suppressed in 0.9% NaCl media and the least suppresed in DMEM media. For example, in 0.9% NaCl media only 0.1 mM of Fe2+/Fe3+ was required to fully suppress the fluorescence of calcein, while in the case of calcein blue, much higher concentrations of iron ions – 1 mM - was required to get the same effect. The results of this work can be useful when estimating the efficiency of cell electropermeabilizationBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Chitin extraction and chitosan production from Chilopoda (Scolopendra cingulata) with identification of physicochemical properties

ISSN: 1555-6107 (electronic version). IF 0.919The Chilopoda is found in a broad array of terrestrial habitats from tropical rain forests to deserts and there are more than 8000 species worldwide. Chitin and chitosan were obtained from Scolopendra cingulata for the first time in this study and physico-chemically characterized. The dry weight chitin content of S. cingulata was found to be 8% of body weight, and it produced chitosan yields of 66%. Analyses results revealed that Chilopoda chitin was in the α-form. The chitosan derived from Chilopoda chitin has a low molecular weight (2.278 kDa). The surface morphology of chitin from S. cingulata consists of dispersed nanopores and nanofibresBiologijos katedraVytauto Didžiojo universiteta

Extraction and characterization of chitins from coackroach ootheca

Currently researchers are isolating chitins from fungal cell walls [1], crustaceans shells [2], insect cuticles [3], sponges [4] and crabs [5] because of its high biomass (chitin value) and large availability. But still there are many organisms which need a thorough examination for their chitin value. Until now none of the studies are reported on physiochemical properties of cockroach egg shells. For the first time egg shells of three notable and abundantly available species of coackroach (Blatta orientalis, Blatella germanica, Periplaneta americana) were physicochemically investigated for its chitin values using FTIR, chitin content %, SEM. For said study selection of these species was made because of its worldwide distribution and large availibility. Periplaneta americana can be easily found in buildings, restaurants, bakeries, basements, sewers, steam tunnels and drainage systems [6]. Blattella germanica is also a widely distributed urban pest. Adult B.germanica is 1/2 to 5/8 inch long and tan to light brown and can found in houses, apartments, restaurants, hotels. Blatta orientalis is often called water bugs because of their damp and cool habitats such as under sinks, washing machines and in damp basements. Adult B.orientalis is about one inch in length. The egg capsule color and size of B.orientalis is dark reddish-brown and 8 to 10 mm respectively. The dry weight chitin content of Blatta orientalis was determined as 0.66%, Blatella germanica 1.4%, Periplaneta americana 6.61%. The SEM analysis showed that in chitins of coackroach egg shells were found highly fibrous, various crumblier clumpy zones and some highly porous zones were detected. After overall results of SEM we can say that surface morphology of coackroach egg shell’s chitin is highly fibrous along with some highly and rarely porous zones. [...]Gamtos mokslų fakultetasVytauto Didžiojo universiteta

Fluctuation in physicochemical properties of chitins extracted from different body parts of honeybee

WOS: 000361184900002PubMed: 26256318It is well known that physicochemical properties of chitin are related with the extraction method. Recently, it was revealed that some physicochemical properties of chitin are also related with taxonomical relationship. For the first time in this study, it was tested how these properties of chitin are affected by different body parts of one organism. The chitins were extracted from five different body parts (head, thorax, abdomen, legs and wings) of honeybee. These chitins were physicochemically characterized and differences among these body parts were identified. Highest chitin content was observed in legs (13.25%) while the lowest from thorax (6.79%). The surface morphologies of the isolated chitin structures from five different body parts were analyzed with SEM, as a result, five different types of surface morphologies were recorded. However, three different types of surface morphologies were observed only in abdomen. Maximum degradation temperatures (DTG(max)) of thorax, abdomen, legs and wings were recorded between 359 and 367 degrees C while DTG(max) value of head chitin was found as 308 degrees C. (C) 2015 Elsevier Ltd. All rights reserved

The influence of medium on Amplex Red as indicator for fluorescent hydrogen peroxide during high-voltage electric impulses

Fluorescent dyes often use to study cell membrane electropermeabilization. When a high-voltage pulse is applied to the electrolyte solution, a variety of electrolysis reactions occur at the metal-electrolyte interfaces. Metal ions that released from the electrodes can react with the fluorescent dyes and quench their fluorescence. This may have an impact when estimating the efficiency of electropermeabilization. In this study, the influence of the medium treated by high-voltage pulse and Al3+, Fe2+, Fe3+, Cr6+, Ni2+ and Mn7+ ions on the fluorescence of fluorescent tracer molecules, including fluorescent indicator for hydrogen peroxide, in various solutions was studied. Cell culture medium consisted of Dulbecco‘s modified Eagle‘s medium supplemented with 9 % fetal bovine serum and 1 % L-glutamine solution (all Sigma–Aldrich Chemie, Steinheim, Germany). 50 μl of the culture medium or a solution of a fluorescent dye was treated with a square-wave electric pulse with the duration of 0,1– 2 ms and the amplitude 0,2–2,4 kV/cm. The fluorescence of calcein, meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS4), doxorubicin (Adriamycin) and H2O2 indicator Amplex Red was studied. The fluorescence was measured at room temperature using Tecan spectrofluorimeter (Tecan Group, Männedorf, Switzerland). The medium which was treated by the electric pulse with the amplitude of 1,2 kV/cm and the duration of 500 and 2000 μs can almost completely quench calcein fluorescence. When the concentration of the metal ions increase in solution, the intensity of the fluorescence of calcein, TPPS4, Adriamycin and Amplex Red decrease. For example, 1 mM of Fe3+ or Ni2+ ions suppressed fluorescence of calcein by 15 and 79 % respectively. Fe3+ at the concentration of 1 mM totally suppressed the fluorescence of porphyrin-sulphonate, by 30 % – the fluorescence of Adriamycin, and by 30–50 % the fluorescence of Amplex Red.[...]Biologijos katedraVytauto Didžiojo universiteta

Fluctuation in physicochemical properties of chitins extracted from different body parts of honeybee

Impact Factor: 4.074It is well known that physicochemical properties of chitin are related with the extraction method. Recently, it was revealed that some physicochemical properties of chitin are also related with taxonomical relationship. For the first time in this study, it was tested how these properties of chitin are affected by different body parts of one organism. The chitins were extracted from five different body parts (head, thorax, abdomen, legs and wings) of honeybee. These chitins were physicochemically characterized and differences among these body parts were identified. Highest chitin content was observed in legs (13.25%) while the lowest from thorax (6.79%). The surface morphologies of the isolated chitin structures from five different body parts were analyzed with SEM, as a result, five different types of surface morphologies were recorded. However, three different types of surface morphologies were observed only in abdomen. Maximum degradation temperatures (DTGmax) of thorax, abdomen, legs and wings were recorded between 359 and 367 °C while DTGmax value of head chitin was found as 308 °CBiologijos katedraVytauto Didžiojo universiteta

Influence of iron ions and hydrogen peroxide on the fluorescence of amplex red dye quenching and CHO cells viability

To study cell membrane electropermeabilization, fluorescent probes is a convenient, sensitive and versatile tool [1]. During high–voltage electric pulses to cell suspension, cell membrane permeabilization and various electrochemical reactions occur at electrode–solution interfaces.These may include evolution of gases (H2, O2, Cl2), changes of pH, dissolution of electrodes (releasing metal ions), formation of hypochloride acid, free radicals, reactive oxygen species (ROS) [2]. Metal ions (iron, chromium, nickel, and manganese), released from the electrodes during a high-voltage pulse, and ROS such as hydrogen peroxide (H2O2) can react with the fluorescent dyes and quench their fluorescence [3, 4]. This may have an impact, when estimating the efficiency of cell electropermeabilization. In this study, the influence of iron ions and hydrogen peroxide on the fluorescence of Amplex Red dye as well as on the viability of CHO cell was studied. With increasing the concentration of the iron ions in the solution the intensity of the fluorescence of Amplex Red decreased. For example, 1 mM of Fe3+ ions suppressed fluorescence of Amplex Red by 30–50%. Also the reduction of the viability of CHO cells by iron ions and hydrogen peroxide has been demonstrated. The results of this work can be useful for optimizing the electroporation methods used in biotechnology, medicine, and food industryBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta